CN102851297B - Myzuspersicae hunchback gene cDNA and application thereof - Google Patents

Abstract

The invention discloses myzuspersicae hunchback gene cDNA and an application thereof; the cDNA of a key gene of hunchback (Mphb) for myzuspersicae embryonic development is transferred to a plant so as to obtain a transgenic plant which expresses Mphb dsRNA and inhibits myzuspersicae reproduction; when a myzuspersicae takes the transgenic tobacco, the expression of Mphb is inhibited; the embryonic development of the myzuspersicae is blocked; the daily mean myzuspersicae producing number is decreased; the reproductive capacity is reduced; and thus the myzuspersicae hunchback gene cDNA can be used for myzuspersicae biological control.

Description

A kind of cigarette aphid hunchback gene cDNA and application thereof

Technical field

The invention belongs to insect molecular biology and plant genetic engineering field, be specifically related to a kind of cigarette aphid fetal development key gene hunchback( mphb) clone of cDNA, express mphbthe acquisition of dsRNA transgene tobacco.

Background technology

Sucking pest is the important Agricultural pests of a class, mainly comprises Homoptera, Hemiptera and part dipteral insect, as planthopper, and rice leafhopper, cotton plant bug and various aphids etc.Sucking pest often infests on spray, leaf, bud, bud, fruit, draws water, robs its nutrition, causes branches and leaves and steamed twisted roll bent, and even whole strain is withered or dead.As planthopper, endanger loss and be 2 to 3 one-tenth, 3 to 5 one-tenth of serious harm losses, even total crop failure.And cotton plant bug has risen to cotton field primary pest in recent years, after transgenic pest-resistant cotton-wadded quilt cotton plant bug is caused harm, cotton buds and bolls come off in a large number, become bell rate obviously to reduce, and output of cotton and quality significantly decline.Sucking pest can also be propagated various virus, and the ability of the aphis propagation viroses of plant occupies the first place of viral vector insect especially, a kind of 107 kind of plant virus diseases of at least can propagating of black peach aphid only, a kind of 55 kind of plant virus diseases of at least can propagating of cotten aphid.The harm that the harm that the viral disease that spreads disease causes often causes considerably beyond aphid itself (Song Xinyuan, 2005).Cigarette aphid ( myzus persicae) be important sucking pest, feeding habits are assorted, and host is extensive, except harm tobacco, also endangers the brassicaceous vegetables such as the fruit trees such as peach, Lee, pears, rape, Chinese cabbage, wild cabbage, leaf mustard, radish and melon, eggplant class and multiple flowers forest.Cigarette aphid generation quantity is large, and harm time is long.If aphid or become aphid with lancet thrust mesophyll, tender stem or tender flower bud, flower, fruit sucks juice, causes dehydration and blade is crispaturaed, be out of shape when serious, poor growth, disorganization is death even.Cigarette aphid also can be propagated the multiple viruses such as cucumber mosaic virus (CMV), tobacco mosaic virus (TMV) (TMV), and agriculture production is caused to heavy losses.

At present, sucking pest control mainly depends on the chemical pesticide with strong action of contace poison and systemic action, as Rogor, Malathion and Deltamethrin etc.Chemical insecticide has been brought into play vital role in insect prevention and control, but life-time service can destroy ecotope, and harm humans is healthy.Conventional breeding has been brought into play important effect in anti-pest crop breed of variety, but the simple conventional breeding means cycle is longer, and is subject to the incompatible restriction of distant hybirdization, and the good anti insect gene of edge species far away is difficult to fast and effeciently be utilized.Some pathogenic micro-organisms can bring out sucking pest prevailing disease, but majority is insect obligate pathogenic fungi, and generally more difficult cultivation and production also exists certain difficulty at present aspect practical application.By the biocontrol fungis such as muscardine, green muscardine fungus and pheromone, phytohemagglutinin control sucking pest, be still in the research and discovery stage, at present domestic not yet have for the high-performance bio agricultural chemicals of sucking pest in land for growing field crops, apply.And natural enemy insect is as the extensive raising of ladybug, aphid cocoon honeybee, Chrysopa etc. with discharge also more difficultly, cost is higher, is difficult in a short time develop into a kind of conventional biological and ecological methods to prevent plant disease, pests, and erosion measure large scale application in agriculture production.

Development along with modern biotechnology, the application of plant genetic engineering in agricultural obtained huge progress, and oneself has the report of the anti-sucking pest of transgenic plant at present, and gene used mainly contains Bt protein gene, protease inhibitor gene, phytohemagglutinin gene.And report, insect has the trend of rising to the tolerance of transgenic plant.

RNA disturbs (RNA Interference, RNAi) be by double-stranded RNA (double stranded RNA, the phenomenon of the homologous mRNA specificity degraded dsRNA) causing, little RNA (siRNA and microRNA) brings into play keying action in RNA interference mechanism.RNA interference is prevalent in plant, animal and fungi, is a kind of monitoring mechanism of resisting poisoning intrusion, suppressing transposon activity, regulate gene expression existing in eukaryote.RNAi technology has broad application prospects in Agricultural pests biological control field.1998, there is scientist's drosophila melanogaster that utilized first RNAi technical study frizzledwith frizzledthe function of 2 genes.Numerous researchs subsequently also show, are injecting or are taking food after target sequence dsRNA, and the expression of insect target gene also can effectively be blocked.By external microinjection dsRNA, can successfully knock out the gene of sucking pest planthopper different expression patterns.Phyllotreta striolata is at injection cross-film odorant receptor genes psOr1after dsRNA, Cruciferae host's Preference is weakened.In artificial diet, add after apysase gene dsRNA, Zea mays root firefly chrysomelid ( diabrotica virgifera virgifera) Triphosaden enzyme gene expression is obviously suppressed, larval growth is stagnated even dead.Bollworm has a P450 full-length gene that induced by gossypol gIP, when P450 full-length gene is expressed in cotton bollworm larvae feed gIPafter the transgene tobacco of dsRNA, in larva midgut tissue gIPtranscriptional level significantly suppressed and followed the rising of catalase activity, simultaneously due in intestines in bollworm gIPthe downward of expressing and larval growth is suppressed, reduces the tolerance of gossypol.In addition, by dsRNA, feed the interference of the endogenous cellulose gene of termite and Hexamerin storage protein gene is had to lethal and teratogenic effect.After dsRNA feeds, silent anopheles costalis's chitinase gene can be by effective silence.Have and report and in paddy rice, express transmembrane transporter gene or carboxypeptidase gene dsRNA can improve the resistance of paddy rice to Nilaparvata lugen (brown planthopper).Except the application in biological control of insect pests, RNAi technology is also the important means of insect functional genome research, be widely used at present the gene regulating research of insect fetal development, but the insect that is mainly limited to sexual propagation is as fruit bat, ostomatid and tiny golden wasp etc.

Gap gene hunchback( hb) coding one contains the transcription factor that zinc refers to, is one of key gene of being responsible in insect fetal development body segment differentiation, research shows that it is at Hymenoptera, Orthoptera, and Diptera, ubiquity in the insects such as Coleoptera, but copy number there are differences. hbmRNA can be provided by parent, also can in zygote, synthesize.Parent hbthe synthetic rear end maternal factor that is subject to of mRNA nanos( nos) regulation and control, and zygote hbmRNA is subject to front end maternal gene bicoid( bcd) control.Fruit bat ( drosophila) in, hbwith orthodenticle( otd) gene is bicoid(bcd)the downstream target gene of gene, bcdwith hbmutual activation otdwith two other front splaying gene empty spiracles( ems) and buttonhead( btd) expression.In addition, hbbe found in the differentiation of fruit bat central nervous system cell and play a significant role.

Insect hbthe silence of gene has strong teratogenesis and lethal effect.Fruit bat hbafunction can cause the disappearance of jaw and chest body segment.By the reticent Tribolium of RNAi triboliumand tiny golden wasp nasoniaparent or zygote hbafter gene, can there is defect in its head and chest.These researchs show fetus growth key gene hbit is a potential target of biological control of insect pests.Insects such as current Hymenoptera (as mosquito), Orthoptera (as locust), Hemipteras (as stinkbug silk worm) hbgene Partial mRNA sequence can be inquired about.Bean aphid ( acyrthosiphon pisum) whole genome sequence announces, bean aphid hb( aphb) gene exists with single copy.But current cigarette aphid hunchback( mphb) be not yet cloned, more not with mphbfor target, preventing and treating the research of cigarette aphid reports.

Summary of the invention

Technical problem to be solved by this invention is: provide a kind of cigarette aphid ( myzus persicae) hunchbackgene ( mphb) cDNA and application thereof, by by cigarette aphid fetal development key gene mphbcDNA forwards in plant, obtains and expresses mphbthe transgenic plant (particularly tobacco) that can suppress the breeding of cigarette aphid of dsRNA, cigarette aphid takes food after transgene tobacco, mphbexpression by inhibitation system, the fetal development of cigarette aphid is obstructed, and per day product aphid number reduces, and fecundity declines, thereby for the biological control of cigarette aphid.

Technical scheme provided by the invention is: a kind of mphbthe cDNA of gene, its nucleotide sequence is as shown in SEQ ID NO.1.This sequence is that contriver clones first from cigarette aphid mphbgene, this gene can, as the target of RNAi, have very large using value in the control of cigarette aphid.

The present invention also provides the albumen of described cDNA coding, and its aminoacid sequence is as shown in SEQ ID NO.2.

A cloning process of described cDNA, comprises the following steps:

(1) from cigarette aphid adult, extract total RNA, then the synthetic cDNA of reverse transcription;

(2) design degenerated primer, described degenerated primer forward sequence is as shown in SEQ ID NO.3, and reverse sequence is as shown in SEQ ID NO.4;

(3) take the cDNA that step (1) obtains is template, with primer step (2) Suo Shu, carries out pcr amplification;

(4) the PCR product that purification step (3) obtains, reclaims object fragment, gets purified product and is connected on pMD18-T carrier and obtains pMD18-T- mphbi, then transforming competent escherichia coli cell Top10, screening is containing pMD18-T- mphbipositive colony;

(5) positive colony of step (4) gained is carried out to sequencing, obtain cDNA claimed in claim 1.

The application of described cDNA: described cDNA is used for preventing and treating a cigarette aphid is chosen 427bp(and is seen 303-729 position Nucleotide in SEQ ID NO.1 from described cDNA sequence) as interfering target, build synthetic mphbthe plant expression vector of dsRNA, obtains expression by Agrobacterium-Mediated Transformation mphbthe transgenic plant that can suppress the breeding of cigarette aphid of dsRNA, cigarette aphid takes food after described transgenic plant, cigarette aphid mphbexpression by inhibitation system, the fetal development of cigarette aphid is obstructed, and fecundity declines.

Above-mentioned application, described plant is tobacco, fruit tree, brassicaceous vegetable, melon or eggplant class.

Above-mentioned application, wherein, obtains and expresses mphbthe method steps of the transgene tobacco that can suppress the breeding of cigarette aphid of dsRNA is as follows:

(1) mphbthe amplification of cDNA fragment target sequence and the introducing of restriction enzyme site: design primer, amplification mphbcDNA fragment 427bp target sequence, introduces respectively in upstream and downstream sali with bamh I site, described primer forward sequence is as shown in SEQ ID NO.5, and reverse sequence is as shown in SEQ ID NO.6;

(2) purification step (1) gained PCR product, reclaims object fragment, gets purified product and is connected on pMD18-T carrier and obtains pMD18-T- mphbi, then transforming competent escherichia coli cell Top10, choosing is containing pMD18-T- mphbipositive colony, cultivate positive colony, therefrom extract pMD18-T- mphbiplasmid;

(3) pMD18-T- mphbienzyme cuts to obtain target fragment, and enzyme is cut after end, reclaims mphbtarget fragment;

(4) enzyme in pUCCRNAi Opposite direction connection site is cut, and enzyme is cut after end, reclaims pUCCRNAi Opposite direction connection fragment;

(5) pUCCRNAi Opposite direction connection fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-R, gets and connects product conversion competent escherichia coli cell Top10, and screening is containing pUCCRNAi- mphbpositive colony of-R;

(6) pUCCRNAi- mphbthe enzyme of-R forward connection site is cut, and enzyme is cut after end, reclaims pUCCRNAi- mphb-R large fragment;

(8) pUCCRNAi- mphb-R large fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-F-R, gets and connects product conversion competent escherichia coli cell Top10, and screening is containing pUCCRNAi- mphbpositive colony of-F-R;

(9) pUCCRNAi- mphb-F-R dsRNA expression cassette mphbthe enzyme of-F-R is cut, and enzyme is cut after end, reclaims pUCCRNAi- mphb-F-R dsRNA expression cassette fragment mphb-F-R;

(10) enzyme of pCAMBIA2300 is cut, and enzyme is cut after end, reclaims pCAMBIA2300 large fragment.

(11) pCAMBIA2300 large fragment with mphbdsRNA expression cassette fragment mphbthe connection of-F-R obtains plant expression vector pCAMBIA2300- mphbi, to get and connect product conversion competent escherichia coli cell Top10, screening is containing pCAMBIA2300- mphbipositive colony; Extract plasmid pCAMBIA2300- mphbi, by enzyme, cut and check order, confirm that vector construction is correct;

(12) plant expression vector pCAMBIA2300- mphbitransform agrobacterium tumefaciens;

(13) acquisition of transgene tobacco:

Adopt leaf disc transformation method to obtain transgenic tobacco plant, step (12) is contained to plant expression vector pCAMBIA2300- mphbiagrobacterium be inoculated in YEB culture medium culturing to OD ₆₀₀=0.6～1.0, contaminate tobacco leaf disc, then leaf dish being transferred to common substratum secretly cultivates, again leaf dish is transferred to division culture medium illumination cultivation 25-35 days, keep 28～30 ℃, differentiate after seedling, seedling is transferred to root media and cultivate, after taking root, transplantation of seedlings is arrived to soil, breeding is to T ₂generation.

The present invention has following beneficial effect:

The present invention clones first from cigarette aphid hunchback( mphb) gene cDNA.And the inventor first with mphbfor interfering target, the RNAi mediating by plant cultivates expression mphbthe transgene tobacco of dsRNA, silence mphbtranscriptional expression, suppress breeding and the population quantity of cigarette aphid, Research foundation is good, perspective height, the risk that insect produces resistance is low, is a kind of anti-good method eliminating aphis.The present invention utilizes cigarette aphid hunchbackthe cDNA of gene expresses by cultivation hunchbackthe tobacco of dsRNA prevents that the method eliminating aphis has dual interference function.Express mphbthe transgene tobacco of dsRNA, after being taken food by cigarette aphid, cigarette aphid mphbexpression by inhibitation system, nervous system development is destroyed, and fetal development is obstructed, and per day product aphid number reduces, and fecundity declines.This kind of transgene tobacco can directly apply to Production of Large Fields, improves the resistance of tobacco to cigarette aphid, reduces the loss causing because of the harm of cigarette aphid, improves yield of tobacco and economic worth.Also this can be grown tobacco and other tobacco bred hybridization, be bred as the tobacco bred of new anti-cigarette aphid, and be finally applied to Production of Large Fields.In addition, the method for the invention controlling object not only comprises the aphid that directly takes food transgene tobacco, also comprises the embryo (offspring) of aphid, and this method can prevent eliminating aphis by double route again like this.Therefore,, although the present invention only interferes target gene for one, there is the feature of dual interference function and dual controlling way.

Assorted due to cigarette aphid feeding habits, host is extensive, except harm tobacco, also endangers the brassicaceous vegetables such as the fruit trees such as peach, Lee, pears, rape, Chinese cabbage, wild cabbage, leaf mustard, radish and melon, eggplant class and multiple flowers forest.Therefore, RNAi plant expression vector pCAMBIA2300-Mphbi carrier can be used for transforming other crop, to obtain other genetically modified crops of expressing Mphb dsRNA, can be used for equally the biological control of cigarette aphid.

Accompanying drawing explanation

Fig. 1 A is cigarette aphid hunchback( mphb) clone of gene and the structure of RNAi plant expression vector.M：250bp ladder marker；1： Mphb cDNA（1325bp）。B is mphbinterfere target sequence amplification.M:DL2000 marker; 1: mphbinterfere target sequence (427bp)

Fig. 2 is that the enzyme of RNAi plant expression vector is cut checking.M: λ- hindiII digest DNA marker; 1:RNAi plant expression vector pCAMBIA2300- mphbiwith pstthe result that I enzyme is cut, shows two bands that expection is big or small; 2: the pCAMBIA2300-cutting without enzyme mphbi.

Fig. 3 is the process of transgene tobacco that obtains by Agrobacterium-Mediated Transformation, with agrobacterium tumefaciens, contaminates tobacco leaf disc, through induction, cultivates altogether, and differentiation, takes root, and transplants the last transgene tobacco that obtains.

Fig. 4 is that the PCR of transgene tobacco detects.Design primer pair aphbdsRNA expression cassette upstream 35S promoter sequence increase, obtain expection 442bp band, explanation aphbthe successful transformation of tobacco of dsRNA expression cassette sequence.M:DL2000 marker; 3,5,12 is three conversion pCAMBIA2300- mphbitransgene tobacco strain; Blank for transforming the transgene tobacco of pCAMBIA2300 empty carrier; Wild-type is the wild-type tobacco of not processing through transgenosis.

Fig. 5 produces the minimizing of aphid number daily after cigarette aphid takes food transgene tobacco.Every strain transgenosis (strain 3,5,12) if 10 new life aphids of tobacco inoculation, the 7-12 days every daily output aphid numbers of statistics, with the tobacco of the wild-type tobacco do not processed through transgenosis and conversion pCAMBIA2300 empty carrier for contrasting.Often grow tobacco and establish 8 repetitions.

Fig. 6 breeds total amount reduction after cigarette aphid takes food transgene tobacco.If 10 new life aphids of every strain transgene tobacco inoculation, a 14th day statistics individual plant cigarette aphid number.

Fig. 7 is that after cigarette aphid takes food transgene tobacco, total biomass reduces.If 10 new life aphids of every strain transgene tobacco inoculation, the cigarette aphid total biomass on the 14th day statistics individual plant tobacco.

Embodiment

Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.

The present invention is cloned into fetal development key gene from cigarette aphid hunchback( mphb) cDNA, it has the sequence shown in SEQ ID NO.1:

(1) information of SEQ ID NO.1

(a) sequence signature:

Sequence length: 1325 base pairs

Type: nucleic acid

Chain: two strands

Topological framework: linearity

(b) molecule type: cDNA

(c) suppose: no

(d) antisense: no

(e) originate at first: cigarette aphid ( myzus persicae)

(f) sequence description: SEQ ID NO.1

(2) information of SEQ ID NO.2

(a) sequence signature

Sequence length: 441 amino acid

Type: amino acid

Chain: strand

Topological framework: linearity

(b) molecule type: protein

(c) sequence description: SEQ ID NO.2

Cigarette aphid of the present invention hunchback( mphb) cloning process of gene cDNA, comprise the following steps:

(1) from cigarette aphid adult, extract total RNA, then the synthetic cDNA of reverse transcription;

(2) according to bean aphid, Hymenoptera, Orthoptera and hemipteran hunchbackthe conserved sequence design degenerated primer of gene:

Mphb forward: 5’-AAAAANCACAARTGCAAACANTG-3’ （SEQ ID NO.3）

Mphb reverse: 5-’CCCATGTGSATMGWGTASAKGA-3’ （SEQ ID NO.4）

(3) take cDNA as template, pcr amplification mphbgene:

PCR condition is: first by following reagent mix together,

Cigarette aphid cDNA:1 μ l

Deoxynucleotide substrate dNTPs:2 μ l

10 * Taq DNA polymerase buffer: 2 μ l

Ex-Taq archaeal dna polymerase: 0.2 μ l

Mphb forward（10μM）： 0.4μl

Mphb reverse（10μM）： 0.4μl

ddH ₂O： 14μl

Cumulative volume: 20 μ l

Reaction conditions is: first 94 ℃ of denaturations are 3 minutes; Then circulate as follows: 94 ℃ 30 seconds, 57.6 ℃ 30 seconds, 72 ℃ 80 seconds, carry out altogether 35 circulations; Last 72 ℃ are extended 10 minutes.As shown in Figure 1A, wherein M is 250bp ladder marker, and No. 1 swimming lane is what expect mphbcDNA sequence (1325bp).

(4) purification step (3) gained PCR product, sepharose reclaims object fragment, gets purified product and is connected on pMD18-T carrier and obtains pMD18-T- mphbi, then transform competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, blue hickie screening is containing pMD18-T- mphbipositive colony;

(5) positive colony of step (4) gained is carried out to sequencing, obtain mphbgene 1325 bp sequences.

Above-mentioned acquisition mphbcDNA fragment is used for cultivating a kind of expression mphbthe transgene tobacco that can suppress the breeding of cigarette aphid of dsRNA, incubation step is as follows:

(1) mphbthe amplification of cDNA fragment target sequence and the introducing of restriction enzyme site.Design primer, amplification mphbcDNA fragment downstream 427bp target sequence (seeing 303-729 position Nucleotide in SEQ ID NO.1), introduces respectively in upstream and downstream sali with bamh I site, primer sequence is as follows:

MphbiF：5’-T GTCGACCAGCCTCAAGCAGCATC-3’（ SalⅠ）（SEQ ID NO.5）

Mphb iR：5’-C GGATCCCGACGAGGAGTTGTTATT-3’（ BamHⅠ）（SEQ ID NO.6）

PCR condition is: first by following reagent mix together,

PMD18-T- mphbiplasmid: 0.5 μ l

Deoxynucleotide substrate dNTPs:2 μ l

10 * Taq DNA polymerase buffer: 2 μ l

Ex-Taq archaeal dna polymerase: 0.2 μ l

Mphb iF（10μM）： 0.4μl

Mphb iR（10μM）： 0.4μl

ddH ₂O： 14.5μl

Cumulative volume: 20 μ l

Reaction conditions is: first 94 ℃ of denaturations are 3 minutes; Then circulate as follows: 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 45 seconds, carry out altogether 35 circulations; Last 72 ℃ are extended 10 minutes.As shown in Figure 1B, wherein M is DL2000 marker, and No. 1 swimming lane is the 427bp of expection mphbtarget sequence.

(2) purification step (1) gained PCR product, sepharose reclaims object fragment, gets purified product and is connected on pMD18-T carrier and obtains pMD18-T- mphbi, then transform competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, blue hickie screening is containing pMD18-T- mphbipositive colony, by liquid LB culture medium culturing for positive colony, from bacterium liquid, extract pMD18-T- mphbiplasmid;

(3) pMD18-T- mphbienzyme cuts to obtain target fragment, and it is as follows that enzyme is cut system:

SalⅠ:3μl

BamHⅠ:3μl

pMD18-T- Mphbi：28μl

10×T buffer: 6μl

At 37 ℃, react 2 hours.

Enzyme is cut after end, and sepharose reclaims mphbtarget fragment.

(4) enzyme in pUCCRNAi Opposite direction connection site is cut, and it is as follows that enzyme is cut system:

SalⅠ:4μl

BamHⅠ:4μl

pUCCRNAi: 26μl

10×T buffer: 6μl

At 37 ℃, react 8 hours.

Enzyme is cut after end, and sepharose reclaims pUCCRNAi Opposite direction connection fragment.

(5) pUCCRNAi Opposite direction connection fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-R, linked system is as follows:

mphbtarget fragment: 6.5 μ l

PUCCRNAi Opposite direction connection fragment: 2 μ l

T4 ligase: 0.5μl

T4 ligase buffer: 1μl

At 16 ℃, react 8 hours

Get and connect product conversion competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, blue hickie screening is containing pUCCRNAi- mphbpositive colony of-R;

(6) pUCCRNAi- mphbthe enzyme of-R forward connection site is cut, and it is as follows that enzyme is cut system:

XhoⅠ:4μl

BglⅡ:4μl

pUCCRNAi: 28μl

10×H buffer: 4μl

At 37 ℃, react 8 hours.

Enzyme is cut after end, and sepharose reclaims pUCCRNAi- mphb-R large fragment.

(8) pUCCRNAi- mphb-R large fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-F-R, linked system is as follows:

mphbtarget fragment: 6.5 μ l

PUCCRNAi- mphb-R large fragment: 2 μ l

T4 ligase: 0.5μl

T4 ligase buffer: 1μl

At 16 ℃, react 8 hours

Get and connect product conversion competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, blue hickie screening is containing pUCCRNAi- mphbpositive colony of-F-R;

(9) pUCCRNAi- hb-F-R dsRNA expression cassette mphbthe enzyme of-F-R is cut, and reaction system is as follows;

pUCCRNAi- Mphb-F-R：34μl

PstⅠ:2μl

10×H buffer: 4μl

At 37 ℃, react 2 hours.

Enzyme is cut after end, and sepharose reclaims dsRNA expression cassette fragment mphb-F-R.

(10) enzyme of pCAMBIA2300 is cut, and system is as follows:

pCAMBIA2300：34μl

PstⅠ:2μl

10×H buffer: 4μl

At 37 ℃, react 2 hours.

Enzyme is cut after end, and sepharose reclaims pCAMBIA2300 large fragment.

(11) pCAMBIA2300 large fragment with mphbdsRNA expression cassette fragment mphbthe connection of-F-R obtains plant expression vector pCAMBIA2300- mphbi, system is as follows:

Mphb-F-R: 6.5μl

PCAMBIA2300 large fragment: 2 μ l

T4 ligase: 0.5μl

T4 ligase buffer: 1μl

At 16 ℃, react 8 hours

Get and connect product conversion competent escherichia coli cell Top10,37 ℃ of dull and stereotyped cultivations, blue hickie screening is containing pCAMBIA2300- mphbipositive colony; Extract plasmid pCAMBIA2300- mphbi, by enzyme, cut and check order, confirm that vector construction is correct.

(12) pCAMBIA2300- mphbienzyme cut checking, it is as follows that enzyme is cut system:

pCAMBIA2300- Mphbi：5μl

PstⅠ:1μl

10×H buffer: 2μl

ddH ₂O：12μl

At 37 ℃, react 2 hours.

Enzyme is cut after end, and sepharose detects enzyme and cuts effect.As shown in Figure 2, wherein M is 250bp ladder marker, and No. 1 swimming lane is the pCAMBIA2300-after enzyme is cut mphbi, show two bands of expecting, the dsRNA expression cassette of plasmid pCAMBIA2300 band and about 1100bp mphb-F-R band, No. 2 swimming lanes are the pCAMBIA2300-cutting without enzyme mphbi.

(12) plant expression vector pCAMBIA2300- mphbitransform agrobacterium tumefaciens

By the freeze-thaw method conversion agrobacterium tumefaciens EHA105 for plant expression vector that cuts through enzyme and check order and detect.Every competent cell (100 μ l) adds pCAMBIA2300- mphbiplasmid 10 μ l, mix, and ice bath 30 min proceed to liquid nitrogen freezing 5 min, put rapidly 37 ℃ of water-bath 5 min.On super clean bench, add 1 ml not containing antibiotic liquid YEB substratum, 28 ℃, 180 r min ^-1on shaking table, cultivate 4 h.By bacterium liquid 5000 r min ^-1centrifugal 5 min, abandoning supernatant, after the resuspended precipitation of the absorption fresh YEB liquid nutrient medium of 100～200 μ l, applying solid YEB substratum is (containing 50 mg L ^-1rifampin and 50 mg L ^-1kantlex), at 28 ℃, cultivate approximately two days, the bacterial plaque growing is inoculated in to liquid YEB substratum (containing 50 mg L ^-1rifampin and 50 mg L ^-1kantlex), in 28 ℃ of shaking tables, 180 r min ^-1cultivate after approximately 1 day and extract plasmid from bacterium liquid, by electrophoresis and PCR, detect, confirm pCAMBIA2300- mphbisuccessfully transform Agrobacterium.

(13) acquisition of transgene tobacco:

Adopt leaf disc transformation method to obtain transgenic tobacco plant.Health tobacco blade is placed in to large culture dish, with 75% alcohol immersion sterilizing 3 min, with aqua sterilisa, rinses 3 times, 0.1% mercuric chloride soaks sterilizing 8 minutes, with aqua sterilisa, rinses 5 times.With the punch tool punching of sterilizing, obtain leaf dish.With inducing culture (MS substratum+0.1 mg L ^-1nAA+1 mg L ^-1bA+3% sucrose+0.375% phytagel, pH, 5.7) 25 ℃ secretly cultivate leaf dish 3～5 days.The Agrobacterium that contains plant expression vector is inoculated in to YEB substratum (5g L ^-1tryptones, 1g L ^-1yeast extract, 5 g L ^-1extractum carnis, 5 g L ^-1sucrose, 0.5 g L ^-1mgSO ₄.7H ₂o, 50 mg L ^-1rifampin, 50 mg L ^-1kantlex), 180～200 r min ^-1, be cultured to OD at 28 ℃ ₆₀₀=0.6～1.0, contaminate leaf dish 30 min.Leaf dish is transferred on sterilizing thieving paper, transferred to common substratum (MS substratum+0.1 mg L after blotting surperficial bacterium liquid ^-1nAA+1 mg L ^-1bA+3% sucrose+0.375% phytagel, pH, 5.7), 25 ℃ of dark cultivations two days in left and right, transfer to division culture medium (MS substratum+0.1 mg L by leaf dish ^-1nAA+1 mg L ^-1bA+3% sucrose+500 mg L ^-1cephamycin+100 mg L ^-1kantlex+0.375% phytagel, pH, 5.7), illumination cultivation, about one month, keeps 28～30 ℃, within every 10 days, changes a division culture medium.Differentiate after seedling, seedling is transferred to root media (MS substratum+0.1 mg L ^-1nAA+3% sucrose+80 mg L ^-1kantlex+250 mg L ^-1cephamycin+0.375% phytagel, pH, 5.7), illumination every day 16 h.After taking root, kalamycin resistance transplantation of seedlings is arrived to soil, breeding is to T ₂generation.As shown in Figure 3, through callus induction, cultivate altogether, differentiation, root culture, has finally obtained Transformation of tobacco, and transplant survival.

(14) PCR of transgene tobacco detects: for the 35S promoter district design primer of dsRNA expression cassette, the transgene tobacco genomic dna of take carries out pcr amplification as template, and the tobacco that transforms empty carrier of take is contrast.Primer sequence is as follows:

35SF：5’-ACAGAACTCGCCGTAAAG-3’ （SEQ ID NO.7）

35SR：5’-AGTGGGATTGTGCGTCAT-3’ （SEQ ID NO.8）

PCR condition is: first by following reagent mix together,

Tobacco DNA:1 μ l

Deoxynucleotide substrate dNTPs:2 μ l

10 * Taq DNA polymerase buffer: 2 μ l

Ex-Taq archaeal dna polymerase: 0.2 μ l

35SF（10μM）： 0.4μl

35SR（10μM）： 0.4μl

ddH ₂O： 14μl

Cumulative volume: 20 μ l

Reaction conditions is: first 94 ℃ of denaturations are 3 minutes; Then circulate as follows: 94 ℃ 30 seconds, 50 ℃ 30 seconds, 72 ℃ 45 seconds, carry out altogether 35 circulations; Last 72 ℃ are extended 10 minutes.After PCR finishes, sepharose detected result.As shown in Figure 4, M:DL2000 marker; 3,5,12 is three conversion pCAMBIA2300- mphbitransgenic line; Blank for transforming the tobacco of pCAMBIA2300 empty carrier; Wild-type is the wild-type tobacco of not processing through transgenosis.Explanation aphbthe successful transformation of tobacco of dsRNA expression cassette sequence.

the anti-aphid test of transgene tobacco

T2, with 10 nascent cigarette aphids of the every strain of writing brush inoculation, connects tobacco and is placed in 20 * 20 * 20(length * wide * height at tobacco top in the 4-5 leaf phase for transgene tobacco after inoculation) in the dependent insect cage of cm with gauze sealing, 25 ℃ of maintenance constant temperature.If 10 new life aphids of every strain transgene tobacco inoculation, often grow tobacco and establish 8 repetitions, the 7-12 days every daily output aphid numbers of statistics.As shown in Figure 5, with wild-type tobacco (wild-type) with the tobacco (blank) that transforms empty carrier for contrasting, take food and contain mphbafter the tobacco of dsRNA expression cassette (strain 3,5,12), cigarette aphid daily output aphid number reduces.

Within after inoculation the 14th day, add up an individual plant cigarette aphid number and cigarette aphid total biomass.As shown in Figure 6, cigarette aphid takes food and contains mphbafter the transgene tobacco of dsRNA expression cassette, breeding total amount reduces.As shown in Figure 7, cigarette aphid takes food and contains mphbafter the transgene tobacco of dsRNA expression cassette, total biomass also significantly reduces.

Claims (2)

1. the application of cDNA in the control of cigarette aphid, its nucleotide sequence of described cDNA is as shown in SEQ ID NO.1, and concrete grammar is as follows: from described cDNA sequence, choose the 427bp of 303-729 position in SEQ ID NO.1 as interfering target, build synthetic cigarette aphid hunchbackthe plant expression vector of the dsRNA of gene, is obtained and is expressed cigarette aphid by Agrobacterium-Mediated Transformation hunchbackthe transgenic plant that can suppress the breeding of cigarette aphid of the dsRNA of gene, cigarette aphid takes food after described transgenic plant, the cigarette aphid of cigarette aphid hunchbackgenetic expression is suppressed, and the fetal development of cigarette aphid is obstructed, and fecundity declines, and wherein said plant is tobacco.

2. application according to claim 1, is characterized in that: obtain and express cigarette aphid hunchbackthe method steps of the transgene tobacco that can suppress the breeding of cigarette aphid of the dsRNA of gene is as follows:

(1) cigarette aphid hunchbackthe amplification of gene cDNA fragment target sequence and the introducing of restriction enzyme site: design primer, amplification cigarette aphid hunchbackgene cDNA fragment 427bp target sequence, introduces respectively in upstream and downstream sali with bamh I site, described primer forward sequence is as shown in SEQ ID NO.5, and reverse sequence is as shown in SEQ ID NO.6;

(2) purification step (1) gained PCR product, reclaims object fragment, gets purified product and is connected on pMD18-T carrier and obtains pMD18-T- mphbi, then transforming competent escherichia coli cell Top10, choosing is containing pMD18-T- mphbipositive colony, cultivate positive colony, therefrom extract pMD18-T- mphbiplasmid;

(3) pMD18-T- mphbienzyme cuts to obtain target fragment, and enzyme is cut after end, reclaims mphbtarget fragment;

(4) enzyme in pUCCRNAi Opposite direction connection site is cut, and enzyme is cut after end, reclaims pUCCRNAi Opposite direction connection fragment;

(5) pUCCRNAi Opposite direction connection fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-R, gets and connects product conversion competent escherichia coli cell Top10, and screening is containing pUCCRNAi- mphbpositive colony of-R;

(6) pUCCRNAi- mphbthe enzyme of-R forward connection site is cut, and enzyme is cut after end, reclaims pUCCRNAi- mphb-R large fragment;

(8) pUCCRNAi- mphb-R large fragment with mphbthe connection of target fragment obtains pUCCRNAi- mphb-F-R, gets and connects product conversion competent escherichia coli cell Top10, and screening is containing pUCCRNAi- mphbpositive colony of-F-R;

(9) pUCCRNAi- mphb-F-R dsRNA expression cassette mphbthe enzyme of-F-R is cut, and enzyme is cut after end, reclaims pUCCRNAi- mphb-F-R dsRNA expression cassette fragment mphb-F-R;

(10) enzyme of pCAMBIA2300 is cut, and enzyme is cut after end, reclaims pCAMBIA2300 large fragment;

(11) pCAMBIA2300 large fragment with mphbdsRNA expression cassette fragment mphbthe connection of-F-R obtains plant expression vector pCAMBIA2300- mphbi, to get and connect product conversion competent escherichia coli cell Top10, screening is containing pCAMBIA2300- mphbipositive colony; Extract plasmid pCAMBIA2300- mphbi, by enzyme, cut and check order, confirm that vector construction is correct;

(12) plant expression vector pCAMBIA2300- mphbitransform agrobacterium tumefaciens;

(13) acquisition of transgene tobacco:

Adopt leaf disc transformation method to obtain transgenic tobacco plant, step (12) is contained to plant expression vector pCAMBIA2300- mphbiagrobacterium be inoculated in YEB culture medium culturing to OD ₆₀₀=0.6～1.0, contaminate tobacco leaf disc, then leaf dish being transferred to common substratum secretly cultivates, again leaf dish is transferred to division culture medium illumination cultivation 25-35 days, keep 28～30 ℃, differentiate after seedling, seedling is transferred to root media and cultivate, after taking root, transplantation of seedlings is arrived to soil, breeding is to T ₂generation.