CN101067137A - Vector with high-efficiency expression of virus gene dsRNA and its application - Google Patents

Abstract

The present invention relates to one kind of vector with high efficiency expression of virus gene double strand RNA (dsRNA). The present invention constructs PBV222 vector in the whole length of 4.1 kb and containing two heat shock promoters PRPL's in opposite direction with PBV220; connects it with cloned virus gene to construct heat shock procaryon expressing vector capable of expressing dsRNA, and transforms to colibacillus with RNaseIII defect for heat induced expression. The cultured fermented liquid after being ultrasonically treated may be used in preventing and treating plant viral diseases. The present invention establishes a cheap efficient dsRNA expressing system, and opens one important way for utilizing RNA silencer in preventing and treating plant viral diseases in agricultural production.

Description

A kind of carrier of high-efficiency expression of virus gene dsRNA and application thereof

Technical field

The present invention relates to a kind of carrier, be specifically related to the carrier of high-efficiency expression of virus gene dsRNA.

Background technology

Plant virus is infected the main diseases original of plant as a class, causes the disease of farm crop, fruit tree, flowers, herbage, medicinal plant in worldwide, causes the decline of yield and quality, even forms crushing harm.According to incompletely statistics, the whole world just accounts for 10% of total output of grain greatly because of the production loss that viral diseases of plants caused every year.But still there is not effective preventing control method on producing at present.

RNA silence (RNA silencing) is meant that the specific interaction based on nucleotide sequence that occurs in rna level comes a kind of regulatory mechanism of inhibition of gene expression, this mechanism extensively is present in the eukaryote, comprise fungi, algae, plant, protozoon, (Agrawal N such as invertebrates and vertebrates, Dasaradhi PVN, Mohmmed A, Malhotra P, Bhatnagar RK, and Mukherjee SK.RNA interference:bilology, mechanism, and applications.Microbiol Mol Biol Rev 2003,67:657-685), also be a kind of general defense response that resists virus infection of plant inherent (Baulcombe D.RNA silencing in plants.Nature 2004,431:356-363).Virus can be induced plant under natural infection RNA silence, and it can be transmitted to by the particular sequence signal and closes on cell (short range) and whole strain plant (long apart from).RNA silence in the plant has wide adaptability, can move and the specific characteristics of sequence.The new plant virus controlling method of design that is found to be of the reticent phenomenon of RNA provides new possibility.

The RNA silence has been widely used in the control of gene functional research, animal virosis and human virus's treatment (Dykxhoorn DM and Lieberman J.Silencing Viral Infection.PLoS Med 2006 at present, 3:1000-1004), the control that is applied to animal virus with the RNA silence is compared, progress based on the plant virus controlling of RNA silence is slow, just causes scientist's attention in recent years.Plant virus and animal virus have a lot of general character mechanism (as reproduction process), use for reference the research method and the system of animal virus, the key issue of utilizing the reticent control of RNA plant virus is studied, be the new approaches of plant virus controlling, also will become the focus (Tenllado F, LlaveC.and Diaz-Ruiz JR.RNA interference as new biotechnological tool for the control ofvirus disease in plants.Virus Res 2004.102:85-96) in plant virus resistance engineering research field.

In recent years, more existing research groups begin to utilize the reticent mechanism of RNA to carry out plant virus resistance research.There are two approach can realize the great expression of dsRNA, thus the startup of the reticent mechanism of inducing plant antiviral rna.1, the transgenic plant of construction expression virus gene dsRNA; 2, the stripped virus gene dsRNA of expressing.Because the transgenic plant of expression dsRNA are marking protein not, this had both improved the disease resistance of transgenic plant, had avoided the misgivings of people to transgenic plant safety again.In theory, any gene on the viral genome can be used for making up the carrier that can express dsRNA and carry out the plant gene conversion, and makes the conversion plant produce resistance to correlated virus by this mechanism.But, utilize construction expression dsRNA carrier to carry out plant gene and transform example that the antiviral transfer-gen plant that obtains produced by successful Application and few because the transformation systems of most of farm crop is immature.And, the dsRNAs of different virus must be changed over to the transgenic plant that host plant could obtain anti-multiple virus, and this is very difficult realization on genetics because crop suffers all generally that multiple virus is compound to infect harm.

Use for reference used external source dsRNA silencer method in the animal, the virus gene dsRNA inducing plant RNA silence that utilizing exsomatizes expresses is prevented and treated virus disease and can be avoided above-mentioned many shortcomings.The prokaryotic expression carrier that present structure can be expressed virus gene dsRNA comprises: 1, make up the carrier that contains this gene inverted repeats; 2, utilize Timmons ﹠amp; Structures such as Fire contain the opposite two-way T7 promotor of both direction carrier L4440 (Specific interference by ingesteddsRNA.Nature, 1998,395:854).All need by the inducing of inductor isopropyl-(IPTG) but two kinds of methods are produced dsRNA, and IPTG costs an arm and a leg, and limits its further popularization in agriculture production.

Therefore making up and a kind ofly can express virus gene dsRNA, need not the prokaryotic expression carrier of inductor again, is to realize that the stripped of virus gene dsRNA efficiently expresses, and induces the gordian technique of host RNA silence.

Summary of the invention

The object of the present invention is to provide a kind of prokaryotic vector of energy high-efficiency expression of virus gene dsRNA, thereby realize utilizing the reticent control of RNA viral diseases of plants.

The technical scheme that realizes the foregoing invention purpose is: the carrier of high-efficiency expression of virus gene dsRNA, be the plasmid that sets out with PBV220, made up the carrier PBV222 that contains the opposite heat-inducible promoter PRPL of both direction, the about 4.1kb of total length, the base sequence of this carrier is shown in SEQ ID N01.

The carrier PBV222 of high-efficiency expression of virus gene dsRNA is used for virus gene dsRNA and in the thermal induction expression method of intestinal bacteria is:

(1) will intend expressing the virogene clone of dsRNA and be connected with the PBV222 carrier, structure can be expressed the heat shock prokaryotic expression carrier of dsRNA;

(2) this prokaryotic expression carrier is transformed the intestinal bacteria specific strain of RNase III defective, cultivate behind the 2h immediately elevated temperature to 42 ℃ for 30 ℃, carry out thermal induction and express, continue to cultivate 4h.

Be described in further detail the present invention below in conjunction with accompanying drawing:

Description of drawings

Accompanying drawing 1 is the building process synoptic diagram of carrier PBV222.

Accompanying drawing 2 is expressed the electrophorogram of the dsRNA that extracts the back for heat-inducible.Wherein swimming lane 1,4 is RNA2 fragment, the MP gene dsRNA that extracts behind the abduction delivering; Swimming lane 2,3 is a blank, and M is the nucleic acid standard.

The present invention extends to external source dsRNA cryptiogene method used in the animal in the plant virus resistance, has proposed utilization The dsRNA control viral diseases of plants that exsomatizes and express has designed a carrier PBV222 that can express dsRNA, this carrier Contain the opposite heat-inducible promoter PRPL of both direction, cut apart by MCS between two heat-inducible promoters, only need plan The viral gene of expressing dsRNA is cloned into the heat shock expression that can realize this gene dsRNA between two heat-inducible promoters, Avoided making up the complex steps of inverted repeat sequence vector. Take this carrier as the basis in conjunction with Escherichia coli HT115 (RNase III defective) (Caenorhabditis Genetics Center, University of Minnesota, Minneapolis, USA) set up disease The external Cheap highly effective expression system of virus gene dsRNA has proposed take the RNA silence as the basis, but has been different from by turning to The novel antiviral strategy of gene expression RNA. The dsRNA spraying plant that utilizes this expression system to produce can effectively prevent and treat The viroses of plant.

1, express the structure of the carrier PBV222 of virus gene dsRNA:

Utilize conventional gene engineering method, with PBV220 (contain the PRPL promoter the High level prokaryotic expression carrier establishment and should With, Zhang Zhiqing etc., viral journal, 1990,6:111-116) be the carrier that sets out, made up and contained the opposite heat of both direction Swash the carrier PBV222 of promoter PRPL.

Concrete steps (seeing Fig. 1) are as follows: the PRPL heat-inducible promoter with pcr amplification PBV220 carrier obtains about 400bp Amplified fragments. The primer of amplification is:

Forward primer 5 '-TACCTGCAGTGTGCTCATACG-3 ',

Reverse primer 5 '-GGATCCTAACCAATGCTTCGTTTC-3 ',

Introduce the PstI restriction enzyme site at 5 ' end, at 3 ' introducing BamHI restriction enzyme site (restriction enzyme site of italicized item for introducing), The pcr amplification condition is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 45 seconds, 56 ℃ of renaturation 30 seconds, 72 ℃ were extended 60 seconds, 35 circulations were extended 10 minutes at last. Use simultaneously PstI and BamHI two in pcr amplified fragment and PBV220 carrier Enzyme is cut, and enzyme is cut rear connection, namely obtains carrier PBV222, the about 4.1kb of this carrier total length.

2, the heat shock construction of prokaryotic expression vector of virus gene dsRNA and conversion are expressed:

(1) virogene that will intend expressing dsRNA is connected with the PBV222 carrier by gene clone, and structure can be expressed the heat shock prokaryotic expression carrier of dsRNA;

(2) with this prokaryotic expression carrier transformed into escherichia coli specific strain HT115 (RNase III defective), cultivate behind the 2h immediately elevated temperature to 42 ℃ for 30 ℃, carry out thermal induction and express, continue to cultivate 4h.

The fermented liquid ultrasonication of cultivating; Simultaneously this fermented liquid is extracted dsRNA with CTAB method ([U.S.] J. Sa nurse Brooker etc., molecular cloning experiment guide (third edition), Science Press, 2002), detect the expression amount of dsRNA;

Handle to express the ferment product of dsRNA with ultrasonic wave (frequency is 20KHZ, handles 15 minutes), smudge cells discharges dsRNA, and this fermented liquid can directly be sprayed and be handled plant and prevent and treat virus disease.

Utilization of the present invention contains the Cheap highly effective expression system of the dsRNA that the intestinal bacteria specific strains of the PBV222 carrier of the opposite heat-inducible promoter PRPL of both direction and RNase III defective sets up, has opened up an important channel for utilize the reticent control of RNA viral diseases of plants in agriculture production.

Embodiment

Embodiment 1:

The stripped expression of utilization cucumber mosaic virus (cucumber mosaic virus, CMV) preparation of the ferment product of RNA2 fragment dsRNA:

1, produces the construction process of the engineering bacteria of dsRNA

(1) CMV PV0419 strain is the extraction of RNA: (DSMZ, band poison blade Germany) adopts silicon grain absorption method to carry out to get inoculation CMV PV0419 strain system.Silicon grain absorption method step is: get the 300mg vegetable material and place the Bioreba plastics to grind bag (Bioreba AG, Switzerland), add 3ml grinding buffer solution (4.0M guanidinium isothiocyanate, 0.2M PH5.2 sodium acetate, 25mM EDTA, 1.0M potassium acetate, 2.5%PVP-40 (w/v), autoclaving 15min is in 4 ℃ of storages) grind.Vegetable material after getting 1ml and grinding adds in the 1.5ml Eppendorf pipe, adds 100 μ l NLS (N-lauryl sarkosyl), puts 70 ℃ and hatches 10min and shake frequently.Put centrifugal 10min (13 behind the ice bath 5min then, 000rpm, RT), get the supernatant liquor 300 μ l after centrifugal, (method according to Rott etc. is prepared to add 25 μ l silicon grains, Rott ME, Jelkmann W.Characterization and detection of several filamentous viruses of cherry:Adaptation of an alternative cloning method (DOP-PCR) and modification of an RNAextraction protocol.Eur J Plant Pathol 2001,107:411-420) 150 μ l100% ethanol and 300 μ l 6M sodium iodide (0.15M S-WATs, the 6M sodium iodide, 4 ℃ of storages) then shook 10 minutes in room temperature.This mixture is removed supernatant behind the centrifugal 1min down in 6000rpm (RT), add 500 μ l lavation buffer solutions (10mM Tris-HCl, pH 7.5,0.5mMEDTA, 5mM sodium-chlor, 50% ethanol (v/v), 4 ℃ of storages) and fully suspend centrifugal 1min.Repeated washing once the back with the 150 μ l distilled waters precipitation that fully suspends, put 70 ℃ hatch behind the 4min in back centrifugal 3min (15,000rpm, RT), it is standby carefully to draw supernatant liquor 130 μ l.

(2) the segmental clone of RNA2 of CMV PV0419 strain system: according to the RNA2 fragment specific PCR amplimer of sequence information design CMV PV0419 strain system, primer sequence is as follows:

Forward primer: 5 '-GATGAATTCYTGTTTGCTCAC-3 ' (Y=C or T)

Reverse primer: 5 '-GGATGGACAACCCGTTC-3 '

The viral RNA that utilizes above-mentioned primer that extraction is obtained carries out the RT-PCR amplification, obtains the RNA2 fragment.Amplification program is 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 45 seconds, and 59 ℃ of renaturation 30 seconds, 72 ℃ were extended 60 seconds, and 35 circulations were extended 10 minutes at last.PCR product 1.0% agarose gel electrophoresis detects, and being presented at 750bp has single band, and is consistent with the expected sequence size;

(3) pcr amplified fragment is utilized restriction enzyme EcoRI enzyme cut, and enzyme is cut the PBV222 expression vector simultaneously, enzyme cuts the PCR product and carrier reclaims respectively after agarose electrophoresis.16 ℃ of connections of T4DNA ligase enzyme are spent the night, PCR fragment orientation is connected on the EcoRI restriction enzyme site of expression vector PBV222, and heat shock transforms ([U.S.] J. Sa nurse Brooker etc., molecular cloning experiment guide (third edition), Science Press, 2002) competent cell HT115 (DE3) (RNase III disappearance).Converted product is coated the LB flat board that contains 150ug/mL AMP and 12.5ug/mL TET, 30 ℃ of incubated overnight.

(4) the positive single bacterium colony of picking, extract plasmid after, the clone's (being recon) who utilizes PCR and EcoRI enzyme to cut to identify correct insertion, further order-checking conclusive evidence.With this expression vector called after PBV222-RNA2, and contain engineering bacteria called after HT115 (the DE3)-PBV222-RNA2 of this expression vector, be the engineering bacteria that RNA2 fragment dsRNA is produced in heat shock of the present invention.

2, the abduction delivering of dsRNA and application

(1) HT115 (containing recombinant plasmid) dilutes 100 times with the 30 ℃ of shaking culture 16h of LB substratum that add 150ug/mL penbritin (AMP) and 12.5ug/mL tetracycline hydrochloride (TET) in 20mL LB substratum, and 30 ℃ of shaking culture grow to OD ₅₉₅=0.5, elevated temperature to 42 ℃ carries out abduction delivering, carries out the extraction of dsRNA behind the shaking culture 4h immediately;

(2) adopt the CTAB method to extract dsRNA: get 2ml bacterium 5000rpm, centrifugal 1min, precipitum adds 500 μ LTE damping fluids and suspends again; Add 30 μ L 10%SDS, mixing, 37 ℃, 1h; Add 100 μ L NaCl (5M), mixing adds the CTAB/NaCl of 80 μ L, mixing, 65 ℃, 10min again; Add equal-volume phenol/chloroform/primary isoamyl alcohol (25: 24: 1) mixed solution, mixing, 4 ℃, the centrifugal 4-5min of 12000rpm; Get the isopropanol precipitating of supernatant with 0.8 volume, 4 ℃, the centrifugal 10min of 12000rpm; Abandon supernatant, the washing with alcohol with 75% (add 500 μ L, 75% ethanol, mix 6000rpm, 4 ℃, centrifugal 5min with wortex device); Be dissolved in ddH after the drying ₂O, electrophoresis detection; (RNase A 1u, the adding final concentration is 0.5moL.L to use DNase (DNase 1u), RNase A digestion respectively ^-1NaCl) after again electrophoresis detection conclusive evidence dsRNA efficiently expressed (see figure 2);

(3) ferment product of abduction delivering is with being directly used in spraying plant leaf control pepper virus disease behind the ultrasonic disruption cell (frequency is 20KHZ, handles 15 minutes).

Embodiment 2:

Utilizing the expression CMV PV0419 strain of exsomatizing is the preparation of the ferment product of CP (capsid protein) gene dsRNA:

(1) design contains the primer in EcoRI site:

Forward primer: 5 '-GAATTCATGGACAAATCTGGATCAC-3 ',

Reverse primer: 5 '-GAATTCCTGGATGGACAACCCG-3 ',

Utilize the PCR method amplification CMV PV0419 strain (DSMZ of system, Germany) CP gene, by conventional molecular cloning means, the CP gene is connected between the opposite two heat-inducible promoter PRPL of PBV222 both direction, structure can be expressed the heat shock prokaryotic expression carrier of dsRNA;

(2) with this prokaryotic expression carrier transformed into escherichia coli specific strain HT115 (RNase III defective), cultivate behind the 2h immediately elevated temperature to 42 ℃ for 37 ℃, carry out thermal induction and express, after continuing to cultivate 4h, fermented liquid can be used for ultrasonication; Simultaneously this fermented liquid is extracted dsRNA with the CTAB method, detect the expression amount of dsRNA;

(3) with the ferment product of ultrasonication expression dsRNA, smudge cells discharges dsRNA, and this fermented liquid can directly be handled plant and prevent and treat virus disease.

Embodiment 3:

Utilizing the expression CMV PV0419 strain of exsomatizing is the preparation of the ferment product of MP (floating preteins) gene dsRNA, and basic skills is the same, and the primer of design is:

Forward primer: 5 '-GAATTCATGGCTTTCCAAGG-3 '

Reverse primer: 5 '-GAATTCACCGTTAACCACCTGCG-3 '

The restriction enzyme site of italicized item for introducing.

＜110〉Huang Heqing

＜120〉a kind of carrier of high-efficiency expression of virus gene dsRNA and application thereof

<160>1

<210>1

<211>4084

<212>DNA

＜213〉full length sequence of PBV222

<220>

<400>1

aattcccggg?gatcctaacc?aatgcttcgt?ttcgtatcac?acaccccaaa?gccttctgct 60

ttgaatgctg?cccttcttca?gggcttaatt?tttaagagcg?tcaccttcat?ggtggtcagt 120

gcgtcctgct?gatgtgctca?gtatcaccgc?cagtggtatt?tatgtcaaca?ccgccagaga 180

taatttatca?ccgcagatgg?ttatctgtat?gttttttata?tgaatttatt?ttttgcaggg 240

gggcattgtt?tggtaggtga?gagatctttt?agctgtcttg?gtttgcccaa?agcgcattgc 300

ataatctttc?agggttatgc?gttgttccat?acaacctcct?tagtacatgc?aaccattatc 360

accgccagag?gtaaaatagt?caacacgcac?ggtgttagat?atttatccct?tgcggtgata 420

gatttaacgt?atgagcacac?tgcagccaag?cttctgtttt?ggcggatgag?agaagatttt 480

cagcctgata?cagattaaat?cagaacgcag?aagcggtctg?ataaaacaga?atttgcctcc 540

cggcagtagc?gcggtggtcc?cacctgaccc?catgccgaac?tcagaagtga?aacgccgtag 600

cgccgatggt?agtgtggggt?ctccccatgc?gagagtagcc?aactgccagg?catcaaataa 660

aacgaaaggc?tcagtcgaaa?gactgggcct?ttcgttttat?ctgttgtttg?tcggtgaacg 720

ctctcctgag?taggacaaat?ccgccgggag?cggatttgaa?cgttgcgaag?caacggcccg 780

gagggtggcg?ggcaggacgc?ccgccataaa?ctgccaggca?tcaaattaag?cagaaggcca 840

tcctgacgga?tggccttttt?gcgtttctac?aaactctttg?tttatttttc?taaatacatt 900

caaatatgta?tccgctcatg?agacaataac?cctgataaat?gcttcaataa?tattgaaaaa 960

ggaagagtat?gagtattcaa?catttccgtg?tcgcccttat?tccctttttt?gcggcatttt 1020

gccttcctgt?ttttgctcac?ccagaaacgc?tggtgaaagt?aaaagatgct?gaagatcagt 1080

tgggtgcacg?agtgggttac?atcgaactgg?atctcaacag?cggtaagatc?cttgagagtt 1140

ttcgccccga?agaacgtttt?ccaatgatga?gcacttttaa?agttctgcta?tgtggcgcgg 1200

tattatcccg?tgttgacgcc?gggcaagagc?aactcggtcg?ccgcatacac?tattctcaga 1260

atgacttggt?tgagtactca?ccagtcacag?aaaagcatct?tacggatggc?atgacagtaa 1320

gagaattatg?cagtgctgcc?ataaccatga?gtgataacac?tgcggccaac?ttacttctga 1380

caacgatcgg?gaggaccgaa?ggagctaacc?gcttttttgc?acaacatggg?ggatcatgta 1440

actcgccttg?atcgttggga?accggatctg?aatgaagcca?taccaaacga?cgagcgtgac 1500

accacgatgc?ctgtagcaat?ggcaacaacg?ttgcgcaaac?tattaactgg?cgaactactt 1560

actctagctt?cccggcaaca?attaatagac?tggatggagg?cggataaagt?tgcaggacca 1620

cttctgcgct?cggcccttcc?ggctggctgg?tttattgctg?ataaatctgg?agccggtgag 1680

cgtgggtctc?gcggtatcat?tgcagcactg?gggccagatg?gtaagccctc?ccgtatcgta 1740

gttatctaca?cgacggggag?tcaggcaact?atggatgaac?gaaatagaca?gatcgctgag 1800

ataggtgcct?cactgattaa?gcattggtaa?ctgtcagacc?aagtttactc?atatatactt 1860

tagattgatt?taaaacttca?tttttaattt?aaaaggatct?aggtgaagat?cctttttgat 1920

aatctcatga?ccaaaatccc?ttaacgtgag?ttttcgttcc?actgagcgtc?agaccccgta 1980

gaaaagatca?aaggatcttc?ttgagatcct?ttttttctgc?gcgtaatctg?ctgcttgcaa 2040

acaaaaaaac?caccgctacc?agcggtggtt?tgtttgccgg?atcaagagct?accaactctt 2100

tttccgaagg?taactggctt?cagcagagcg?cagataccaa?atactgtcct?tctagtgtag 2160

ccgtagttag?gccaccactt?caagaactct?gtagcaccgc?ctacatacct?cgctctgcta 2220

atcctgttac?cagtggctgc?tgccagtggc?gataagtcgt?gtcttaccgg?gttggactca 2280

agacgatagt?taccggataa?ggcgcagcgg?tcgggctgaa?cggggggttc?gtgcacacag 2340

cccagcttgg?agcgaacgac?ctacaccgaa?ctgagatacc?tacagcgtga?gcattgagaa 2400

agcgccacgc?ttcccgaagg?gagaaaggcg?gacaggtatc?cggtaagcgg?cagggtcgga 2460

acaggagagc?gcacgaggga?gcttccaggg?ggaaacgcct?ggtatcttta?tagtcctgtc 2520

gggtttcgcc?acctctgact?tgagcgtcga?tttttgtgat?gctcgtcagg?ggggcggagc 2580

ctatggaaaa?acgccagcaa?cgcggccttt?ttacggttcc?tggccttttg?ctggcctttt 2640

gctcacatgt?tctttcctgc?gttatcccct?gattctgtgg?ataaccgtat?taccgccttt 2700

gagtgagctg?ataccgctcg?ccgcagccga?acgaccgagc?gcagcgagtc?agtgagcgag 2760

gaagcggaag?agcgccctta?tctttccctt?tatttttgct?gcggtaagtc?gcataaaaac 2820

cattcttcat?aattcaatcc?atttactatg?ttatgttctg?aggggagtga?aaattcccct 2880

aattcgatga?agattcttgc?tcaattgtta?tcagctatgc?gccgaccaga?acaccttgcc 2940

gatcagccaa?acgtctcttc?aggccactga?ctagcgataa?ctttccccac?aacggaacaa 3000

ctctcattgc?atgggatcat?tgggtactgt?gggtttagtg?gttgtaaaaa?cacctgaccg 3060

ctatccctga?tcagtttctt?gaaggtaaac?tcatcacccc?caagtctggc?tatgcagaaa 3120

tcacctggct?caacagcctg?ctcagggtca?acgagaatta?acattccgtc?aggaaagctt 3180

ggcttggagc?ctgttggtgc?ggtcatggaa?ttaccttcaa?cctcaagcca?gaatgcagaa 3240

tcactggctt?ttttggttgt?gcttacccat?ctctccgcat?cacctttggt?aaaggttcta 3300

agcttaggtg?agaacatccc?tgcctgaaca?tgagaaaaaa?cagggtactc?atactcactt 3360

ctaagtgacg?gctgcatact?aaccgcttca?tacatctcgt?agatttctct?ggcgattgaa 3420

gggctaaatt?cttcaacgct?aactttgaga?atttttgcaa?gcaatgcggc?gttataagca 3480

tttaatgcat?tgatgccatt?aaataaagca?ccaacgcctg?actgccccat?ccccatcttg 3540

tctgcgacag?attcctggga?taagccaagt?tcatttttct?ttttttcata?aattgcttta 3600

aggcgacgtg?cgtcctcaag?ctgctcttgt?gttaatggtt?tcttttttgt?gctcatacgt 3660

taaatctatc?accgcaaggg?ataaatatct?aacaccgtgc?gtgttgacta?ttttacctct 3720

ggcggtgata?atggttgcat?gtactaagga?ggttgtatgg?aacaacgcat?aaccctgaaa 3780

gattatgcaa?tgcgctttgg?gcaaaccaag?acagctaaaa?gatctctcac?ctaccaaaca 3840

atgcccccct?gcaaaaaata?aattcatata?aaaaacatac?agataaccat?ctgcggtgat 3900

aaattatctc?tggcggtgtt?gacataaata?ccactggcgg?tgatactgag?cacatcagca 3960

ggacgcactg?accaccatga?aggtgacgct?cttaaaaatt?aagccctgaa?gaagggcagc 4020

attcaaagca?gaaggctttg?gggtgtgtga?tacgaaacga?agcattggtt?aaaaattaag 4080

gagg 4084

。

Claims (2)

1, a kind of carrier of high-efficiency expression of virus gene dsRNA is characterized in that this carrier is the plasmid that sets out with PBV220, has made up the carrier PBV222 that contains the opposite heat-inducible promoter PRPL of both direction, and the base sequence of this carrier is shown in SEQ ID NO1.

2, a kind of application of carrier of high-efficiency expression of virus gene dsRNA as claimed in claim 1 is characterized in that this carrier is used for virus gene dsRNA and in the thermal induction expression method of intestinal bacteria is:

(1) will intend expressing the virogene clone of dsRNA and be connected with the PBV222 carrier, structure can be expressed the heat shock prokaryotic expression carrier of dsRNA;

(2) this prokaryotic expression carrier is transformed the intestinal bacteria specific strain of RNase III defective, cultivate behind the 2h immediately elevated temperature to 42 ℃ for 30 ℃, carry out thermal induction and express, continue to cultivate 4h.

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