CN105420244A - cDNA total-length sequence of Chouioia cunea heat shock protein Hsp90 and application - Google Patents

Abstract

The invention discloses a cDNA total-length sequence of a Chouioia cunea heat shock protein Hsp90 and application. The total length of the sequence is 2169 bp, and the sequence contains a complete open reading frame and is encoded with 722 amino acids. By the adoption of a real-time fluorescent quantitative PCR method, Chouioia cunea Hsp90 gene expression changes at the different temperatures are researched, results show that both high temperature and low temperature stress induce increase of the Chouioia cunea Hsp90 expression quantity, and the expression quantities at the temperature of 40 DEG C and -7 DEG C are maximum. The cDNA total-length sequence can provide a molecular basis for scientific utilization of the natural enemy insects Chouioia cunea.

Description

Zhou Shi nibbles cDNA full length sequence 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); The subsidy of Tianjin Normal University doctor fund (52XB1003) and Tianjin animals and plants resistance key lab open fund.

Technical field

The invention belongs to field of molecular biotechnology, research Zhou Shi nibbles the change that Hsp90 under the cDNA full length sequence of chalcid fly heat shock protein Hsp90 and differing temps expresses.

Background technology

Heat shock protein Heatshockprotein (Hsp) is that a class extensively exists in vivo, the high conservative property protein very responsive to inside and outside environmental change, the various physiological metabolism approach of wide participation, has become a focus in biological research fields in recent years.Large I according to its molecular weight is divided into different protein families, wherein Hsp90 and temperature stress irritability closely related.Temperature affects organism to survive vital environmental factor, and only in certain temperature range, organism just can carry out material energy metabolism to maintain normal physiological activity, and it is even dead that the tolerance range exceeding temperature all can make biological growing stagnate.In general after organism is subject to high temperature or low temperature stimulation, albumen resultant quantity can have significant change, in order to strengthen degeneration-resistant border ability.Due to heat shock protein can coerce eliminate after keep certain content that organism is better survived in for some time, can think that it exists and organism is conformed important role.

Zhou Shi nibbles chalcid fly chouioiacuneayang is the important invasive species fall webworms of China hyphantriacunea(Drury) important parasite, the long 1.1-1.5mm of body, troops endoparasitism in fall webworms pupa, is the Natural Enemies factor suppressing fall webworms, plays an important role to the harm controlling fall webworms.Except parasitizing fall webworms, also can parasitize the Chrysomelidae and ladybirds etc. of lepidopterous Lasiocampidae, Lymantriidae, Notodontidae, Geometridae, diamond-back moth section and dipterous Nuscidae, Larvaevoridae and Coleoptera

Discharged by field c.cuneathe object of effectively control fall webworms can be reached.But, under lowered in field environment, c.cuneavigor can be subject to the impact of multiple factors, wherein, temperature, as a main ecological factor, can affect c.cuneavitality .throw in chalcid fly in suitable temperature range, just can play better prevention effect.The present invention is checked order by transcript profile, reports the cDNA full length sequence that a Zhou Shi nibbles chalcid fly heat shock protein Hsp90, and this sequence can be the temperature tolerance mechanism that further investigation Zhou Shi nibbles chalcid fly and lays the foundation.By real time fluorescence quantifying PCR method, the change of Chouioia cunea Hsp90 genetic expression under research differing temps, result of study can be scientific utilization natural enemy insect Zhou Shi and nibbles chalcid fly and provide fundamental basis.

Summary of the invention

The object of the invention is the cDNA complete sequence that report Zhou Shi nibbles chalcid fly heat shock protein gene Hsp90 gene and the impact that temperature association is compeled thereof.

The technical solution used in the present invention obtains the complete sequence that Zhou Shi nibbles chalcid fly heat shock protein gene Hsp90 gene, the change of Hsp90 genetic expression under the technique study differing temps passing through RT-qPCR by transcript profile order-checking.

The invention discloses following technology contents for this reason:

Zhou Shi nibbles the nucleotide sequence of chalcid fly heat shock protein Hsp90, this sequence 2169bp, containing complete open reading frame, has the nucleotide sequence shown in SEQIDNO.1.Corresponding aminoacid sequence is as shown in SEQIDNO.2; This sequence encoding 722 amino acid, molecular weight is 83.3KDa, and iso-electric point is 5.01.

The present invention further discloses nucleotides sequence that Zhou Shi nibbles chalcid fly heat shock protein Hsp90 and be listed in application for detecting the sick aspect of control fall webworms.Described control fall webworms refer to: Hsp90 genetic expression is by temperature adjusting, by detecting the expression of Hsp90 gene, can learn that Zhou Shi nibbles the tolerance situation of chalcid fly for temperature stress, discharge chalcid fly nibbling lower than Zhou Shi under chalcid fly bears ultimate temperature, better can play the object of control fall webworms.By real time fluorescence quantifying PCR method, the change of Chouioia cunea Hsp90 genetic expression under research differing temps, result of study can be scientific utilization natural enemy insect Zhou Shi and nibbles chalcid fly and provide fundamental basis.

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

Table 1

Table 2

MPADVAMDSTEVETFAFQAEIAQLMSLIINTFYSNKEIFIRELISNSSDALDKIRYESLTDPSKLDSCKELLIKMIPNKNDRTLTFIDSGIGMTKADLVNNLGTIAKSGTKAFMEALQAGADISMIGQFGVGFYSAYLVADKVTVVSKHNDDEQYIWESSAGGSFTVRPDKGEPLGRGTKIILHIKEDQAEYLEESKIKEIVKKHSQFIGYPIKLVLEKERDKELSEDEEEDDKKDEDKEEDTEKPKIEDVGEDEDEEKSKEEKKKKKKTVKEKYTDEEELNKTKPIWTRNPDDITHEEYGEFYKSLTNDWEDHLAVKHFSVEGQLEFRALLFIPRRAPFDLFENKKRKNNIKLYVRRVFIMDNCEDLIPEYLNFIRGVVDSEDLPLNISREMLQQNKILKVIRKNLVKKCLELFEELAEDKENYKKCYEQFSKNLKLGIHEDSTNRKKLSELLRYHTSASGDEQCSLKDYVGRMKENQKHIYYITGESKDQVANSAFVERVRKRGFEVVYMTEPIDEYVVQQLKEFDGKQLVSVTKEGLELPVDEDEKKKMEEDKTKFENLCKVMKDILDKRVEKVVVSNRLVDSPCCIVTSQYGWTANMERIMKAQALRDTSTMGYMAAKKHLEINPDHPIMDNLRLKAEADKHDKSVKDLVMLLFETALLSSGFSLEDPGVHASRIYRMIKLGLGLDDEEMAVEEEKTENEVPPLEGDAEEASRMEEVD*

The advantage that Zhou Shi disclosed by the invention nibbles the cDNA full length sequence of chalcid fly heat shock protein Hsp90 is:

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

(1) change of Hsp90 genetic expression under the technique study differing temps passing through RT-qPCR, the expression level that result is presented at the Hsp90 gene that condition of different temperatures Xia Zhoushi nibbles in chalcid fly body is in a dynamic process, high temperature and low temperature stress all make Zhou Shi nibble Hsp90 in chalcid fly body a high expression amount, 40 DEG C and-7 DEG C of expression in vivo amounts maximum.Therefore proposition of the present invention have studied the change that Zhou Shi nibbles chalcid fly adaptation external environment; avoid the protection mechanism of the injury being forced factor pair self; control fall webworms are of great importance, for green control forestry pest provides a new thinking and technology.

Accompanying drawing explanation

Fig. 1 be Zhou Shi nibble chalcid fly Hsp90 same time differing temps coerce lower expression change.

Embodiment

Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim; Wherein agents useful for same all has commercially available.The extraction of the extraction Chouioia cunea total serum IgE described in the present invention, the synthesis of cDNA, the methods such as real time fluorescent quantitative RT-qPCR are all this area proven technique, and test kit TransScript RT, TransStart TopGreenqPCRSuperMix etc. can buy from manufacturer.

Embodiment 1:

Obtain Chouioia cunea Hsp90 gene order

Experiment material takes from the Chouioia cunea (culture condition: in growth cabinet (PQX-350H), temperature 25 DEG C, relative humidity 60% ~ 80%, unglazed dark) of indoor Secondary Culture.The female Zhou Shi got after sprouting wings in 24h nibbles chalcid fly, cuts feeler, be dipped in RNAlater(Ambion company immediately, AM7020 under anatomical lens) in; The feeler of every 200 female Chouioia cuneas is stored in a 1.5mL centrifuge tube, collect 8 pipe samples altogether, and at-20 DEG C cryopreservation, send Hua Da Gene science Services Co., Ltd (http://www.genomics.cn/index) to carry out transcript profile order-checking after sample preparation completes.

Application high-flux sequence platform IlluminaHiSeq 2000 couples of Zhou Shi nibble chalcid fly feeler sample and carry out transcript profile order-checking, unigene is spliced into after adopting Trinity software to carry out cluster to each reading sequence fragment, the judgement of target sequence cDNA total length is carried out again in conjunction with bioinformatics software, carry out the retrieval of Blast homologous sequence to add their confirmation, obtain the sequence of Hsp90 gene cDNA sequence and coded amino acid.

Embodiment 2:

The extraction of total serum IgE

Choose about 30 Chouioia cuneas and carry out treatment of different temperature, pyroprocessing (1h) is 28 DEG C, 32 DEG C, 36 DEG C, 40 DEG C and 42 DEG C; Subzero treatment (1h) is 11 DEG C, 6 DEG C, 1 DEG C ,-3 DEG C ,-7 DEG C.Process after product use immediately or be stored in-20 DEG C freezing.

(1) getting about 30 Chouioia cuneas is placed in the sterile centrifugation tube of 1.5mL, pours liquid nitrogen into and rapid grinding rod fully grinds.600 μ LTrizon solution are added centrifuge tube, leaves standstill 5min.

(2) 200 μ L chloroforms are added centrifuge tube, thermal agitation 15s, leave standstill 3min.

The centrifugal 15min of 12000rpm at (3) 4 DEG C, proceeds to colourless for upper strata aqueous phase in new sterile centrifugation tube, adds 400 μ L Virahols, places 20min at-20 DEG C, the centrifugal 10min of 12000rpm.

(4) carefully outwell liquid in pipe, retain precipitation, add 600 μ L75% washing with alcohol precipitations.The centrifugal 5min of 7500rpm at 4 DEG C, then repeat a washing with alcohol precipitation operation.

(5) careful abandoning supernatant, drying at room temperature 2min.RNA precipitation is dissolved in 20 μ LRelutionBuffer, if desired can 55 DEG C-60 DEG C water-bath 10min.

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

Embodiment 3:

The synthesis of cDNA

Using Chouioia cunea total serum IgE described in embodiment 2 as template.Get the 0.2mL centrifuge tube of a sterilization, add following reaction system (20 μ L system): TotalRNA5 μ L, orRandomPrimer(N9) (0.1 μ g/ μ L) 1 μ L, 2 × TSReactionMix10 μ L, TransScript RT/RIEnzymeMix1 μ L, RNase-freeWater3 μ L, mixing.

Reaction conditions is 25 DEG C of water bath heat preservations 10 minutes, 42 DEG C of water bath heat preservations 30 minutes, and 85 DEG C of heat 5 minutes, lose activity to make the TransScript RT retained in centrifuge tube.Product use immediately or be stored in-20 DEG C freezing.

Embodiment 4:

Quantitative fluorescent PCR reaction system and condition

(1) design of primers: according to the full length sequence of gained Hsp90 gene, use Primer5 software design to be applicable to the Auele Specific Primer of real-time PCR detection, primer sequence is as follows:

(2) real-time fluorescence PCR: use SYBRGreen chimeric fluorescent method to carry out RT-PCR analysis.CF96XPCR instrument (Bio-Rad) is used to carry out PCR reaction.With the cDNA of synthesis in above-described embodiment 3 for template, with Auele Specific Primer designed in above-mentioned (1), carry out real-time fluorescent PCR amplification reaction, 3 parallel pipes established by each sample, and after amplification, the Ct value (the reaction cycle number experienced when the fluorescent signal namely in each reaction tubes arrives the thresholding of setting) of gained 3 parallel pipes is taken the mean.

Real-time fluorescent PCR amplification system arranges as follows:

Real-time fluorescent PCR amplification optimum configurations is as follows:

50℃120s；

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

Chouioia cunea at different temperatures Hsp90 gene relative expression quantity calculates:

When the amplification efficiency of goal gene and reference gene is close, adopt 2 ^{-Δ Δ CT}method calculates the mRNA relative expression quantity of Hsp90 gene at each temperature, carries out one-way analysis of variance with SPSS19.0 software, the significance of difference between DuncanShi duncan's new multiple range method inspection different treatment.

Fig. 1 is the relative expression quantity that differing temps Xia Zhoushi nibbles chalcid fly Hsp90 gene.No matter be the expression that high temperature or low temperature can affect that Zhou Shi nibbles the Hsp90 in chalcid fly body as seen from the figure.When low temperature stress, along with the continuous reduction of temperature, Zhou Shi nibbles the relative expression quantity of the Hsp90 in chalcid fly body in the trend risen, and reaches maximum expression amount-7 DEG C time; When high temperature stress, along with the continuous rising of temperature, Zhou Shi nibbles the relative expression quantity of the Hsp90 in small peak body also in the trend risen, but reaches maximum expression amount 40 DEG C time.For later which kind of external temperature transferring style of calligraphy characterized by hollow strokes moth Zhou Shi in, to nibble chalcid fly control fall webworms significant for this.

SEQUENCELISTING

<110> Tianjin Normal University

<120> Zhou Shi nibbles cDNA full length sequence and the application of chalcid fly heat shock protein Hsp90

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<170>PatentInversion3.5

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<211>2169

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aacttgggtacaattgcaaaatcaggcacaaaagctttcatggaagctctgcaggctgga360

gctgatatatctatgattggccagtttggcgttggtttctactctgcttaccttgttgcc420

gacaaagtcactgttgtctcgaaacataacgatgatgaacaatacatctgggaatcatcg480

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aaagaagaaaagaagaaaaagaagaagactgtgaaagagaaatacacagatgaagaggaa840

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ggtgaattctacaagagcttgacaaatgactgggaagaccatcttgctgtcaaacacttt960

tcagttgagggtcagcttgagttccgtgctcttcttttcatccctcgccgggcacctttt1020

gatctatttgaaaacaagaagagaaagaacaatatcaaactctacgtgagaagggtcttc1080

attatggacaactgtgaggaccttatcccagagtatttgaacttcatccgtggtgtggta1140

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aaagtcatcaggaagaatcttgtcaaaaaatgcttggaactctttgaagagctagctgaa1260

gacaaagaaaactacaagaaatgctacgagcaatttagcaagaaccttaagcttggtatc1320

catgaagatagcaccaatagaaagaagctctctgaacttctgcgttatcacacatctgct1380

tctggcgatgaacagtgctctctgaaagactatgttggacgtatgaaagaaaatcagaaa1440

cacatttattacatcactggcgagagcaaagatcaggtagcgaacagcgcctttgtcgag1500

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gtcgactaa2169

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<213> Zhou Shi nibbles the aminoacid sequence of chalcid fly Hsp90 genes encoding

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MetProAlaAspValAlaMetAspSerThrGluValGluThrPheAla

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PheGlnAlaGluIleAlaGlnLeuMetSerLeuIleIleAsnThrPhe

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TyrSerAsnLysGluIlePheIleArgGluLeuIleSerAsnSerSer

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AspAlaLeuAspLysIleArgTyrGluSerLeuThrAspProSerLys

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LeuAspSerCysLysGluLeuLeuIleLysMetIleProAsnLysAsn

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AspArgThrLeuThrPheIleAspSerGlyIleGlyMetThrLysAla

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AspLeuValAsnAsnLeuGlyThrIleAlaLysSerGlyThrLysAla

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PheMetGluAlaLeuGlnAlaGlyAlaAspIleSerMetIleGlyGln

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PheGlyValGlyPheTyrSerAlaTyrLeuValAlaAspLysValThr

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ValValSerLysHisAsnAspAspGluGlnTyrIleTrpGluSerSer

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AlaGlyGlySerPheThrValArgProAspLysGlyGluProLeuGly

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ArgGlyThrLysIleIleLeuHisIleLysGluAspGlnAlaGluTyr

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IleGlyTyrProIleLysLeuValLeuGluLysGluArgAspLysGlu

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LeuSerGluAspGluGluGluAspAspLysLysAspGluAspLysGlu

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GluAspThrGluLysProLysIleGluAspValGlyGluAspGluAsp

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GluGluLysSerLysGluGluLysLysLysLysLysLysThrValLys

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GluLysTyrThrAspGluGluGluLeuAsnLysThrLysProIleTrp

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LysValIleArgLysAsnLeuValLysLysCysLeuGluLeuPheGlu

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SerLysAsnLeuLysLeuGlyIleHisGluAspSerThrAsnArgLys

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ValValValSerAsnArgLeuValAspSerProCysCysIleValThr

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SerGlnTyrGlyTrpThrAlaAsnMetGluArgIleMetLysAlaGln

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AlaLeuArgAspThrSerThrMetGlyTyrMetAlaAlaLysLysHis

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GlyValHisAlaSerArgIleTyrArgMetIleLysLeuGlyLeuGly

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LeuAspAspGluGluMetAlaValGluGluGluLysThrGluAsnGlu

690695700

ValProProLeuGluGlyAspAlaGluGluAlaSerArgMetGluGlu

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ValAsp

Claims (4)

1. Zhou Shi nibbles the nucleotide sequence of chalcid fly heat shock protein Hsp90, this sequence 2169bp, containing complete open reading frame, has the nucleotide sequence shown in SEQIDNO.1.

2. sequence according to claim 1, aminoacid sequence corresponding to wherein said nucleotide sequence is as shown in SEQIDNO.2; This sequence encoding 722 amino acid, molecular weight is 83.3KDa, and iso-electric point is 5.01.

3. the nucleotides sequence that Zhou Shi described in claim 1 nibbles chalcid fly heat shock protein Hsp90 is listed in the application for detecting control fall webworms aspect.

4. application according to claim 3, wherein said control fall webworms refer to: Hsp90 genetic expression is by temperature adjusting, by detecting the expression of Hsp90 gene, can learn that Zhou Shi nibbles the tolerance situation of chalcid fly for temperature stress, discharge chalcid fly nibbling lower than Zhou Shi under chalcid fly bears ultimate temperature, better can play the object of control fall webworms.