CN103993033B - Dual anti-iris cymbidium mosaic virus and the RNAi carrier of odontoglossum ring spot virus and application thereof - Google Patents

Abstract

The invention belongs to biology field, the present invention relates to a kind of dual anti-iris cymbidium mosaic virus and the RNAi carrier of odontoglossum ring spot virus, and its application in mediating the resistance to iris cymbidium mosaic virus (CymMV) and odontoglossum ring spot virus (ORSV).The dual anti-iris cymbidium mosaic virus of the present invention and the RNAi carrier of odontoglossum ring spot virus, its sequence is as shown in Seq ID No:1.The present invention is applied to the iris new varieties by the dual anti-two-strain of gene transformation technology selection-breeding, can effectively preventing iris produce on cymbidium mosaic virus and odontoglossum ring spot virus, produce huge economic benefit.

Description

Dual anti-iris cymbidium mosaic virus and the RNAi carrier of odontoglossum ring spot virus and application thereof

Technical field

The invention belongs to biology field, the present invention relates to a kind of dual anti-iris cymbidium mosaic virus and tooth is blue The RNAi carrier of ring spot virus, and its mediation to iris cymbidium mosaic virus (CymMV) and tooth orchid ring speckle Application in the resistance of virus (ORSV).

Background technology

Iris (Phalaenopsis ssp.) is the orchid family Phalaenopsis flowers, is found the earliest in 1750, so far Have been found that more than 70 initial species, on TaiWan, China and Thailand, Philippine, Malaysia, Indonesia and other places All there is distribution.Iris is graceful because of its flower-shape, and color is magnificent, for the treasure in hot bandwidth, has " queen in orchid " Good reputation, add storage tolerance, the advantage such as be suitable for indoor placement, economic benefit high becomes the new lover of flower industry, butterfly Phalaenopsis potted flower has become the agricultural industry that the whole world is important.China's iris production development in recent years is rapid, and 1997 big Land iris manufacturing enterprise cans be counted on one's fingers, year landings only have about 200,000 strains, and to iris landings in 2003 More than 1,0,000,000 strains, the output value reaches 500,000,000 yuan, 2010, and China's iris annual flower landings have reached 2,610 Wan Zhu.

Along with the implant mass of China's iris, disease is also on the rise.Iris virosis is on iris An important disease of class, mostly rely on tissue culture to carry out asexual propagation on producing due to iris, virus is in battalion Supporting in organ and constantly accumulate, virus also can be carried out by the wound that plant management in tissue culture and greenhouse causes simultaneously Propagate, make iris prevention and cure of viruses difficulty, have a strong impact on the states such as growth and the ornamental quality of iris, and America and Europe Family is very strict to the quarantine of iris virus, and outlet iris seedling and finished product with virosis are looked forward to China's export Industry brings about great losses.Also do not have effective medicine to carry out preventing and treating iris virosis on producing at present, can only pass through Eliminate diseased plant and Shoot Tip Culture detoxification prevents and treats, but Shoot-tip Grafting In Vitro operation easier is big, expends substantial amounts of manpower Material resources, detoxification efficiency is the most unsatisfactory, and also can infect virus in expanding propagation and cultivation, hence with The technology selection-breeding iris disease-resistant varieties such as genetic engineering become in production in the urgent need to.

At present, the orchid virus the most reported has: cymbidium mosaic virus, odontoglossum ring spot virus, sword-leaved cymbidium are light Property mosaic virus, cybidium ring spot virus, annulus zonatus, all ages clover mosaic virus, Herba Dendrobii mosaic virus, About 10 kinds of viruses such as cattleya mottle virus and cucumber mosaic virus, wherein with cymbidium mosaic virus (Cymbidium Mosaic virus, CymMV) and odontoglossum ring spot virus (Odontoglossum ringspot virus, ORSV) generation The most universal.Cymbidium mosaic virus and odontoglossum ring spot virus are the two kinds of main virus causing diseases infecting iris.

CymMV is subordinate to closterovirus section, potexvirus, long 475nm～490nm of threadlike particle, diameter 13nm. ORSV is subordinate to Tobamovirus, the long 300nm of shaft-like particle, diameter 18nm.Two-strain can individually infect Or Combined Infection orchid, cause the symptoms such as floral leaf, necrosis, vein chlorisis, streak, can lead during Combined Infection The serious symptoms such as plant is downgraded in cause, leaf malformation, cause the multiple heat such as iris, Herba Dendrobii orchid, Cymbidium hyridus, oncidiumLuridum Band is blue and state orchid produces serious plant disease, is substantially reduced the commodity value of Cymbidium ensifolium (L.) Sw..China's report orchid virus starts from 1983 Year Zhu Benming etc. is separated to two-strain from the Cymbidium ensifolium (L.) Sw. in Shanghai, is accredited as cymbidium mosaic virus and tooth orchid ring spot Poison.Shen Shulin (1990) to both virus biology, the further systematic study of biochemical characteristic, it was demonstrated that be China Topmost virus on Cymbidium ensifolium (L.) Sw..

Iris virus is main to be propagated by amboceptor or juice.Amboceptor is important route of transmission, can pass through aphid, Nematicide, demodicid mite class etc. are propagated.It is the phase mutual friction by blade that juice is propagated, or the tissue culture of people and field Operation, makes healthy leaves cause microtrauma, causes virus disseminating.Iris is made to infect virus by above two approach, And reach next asexual generation by tissue culture expanding propagation, thus accumulate for a long time in iris trophosome, cause yield and Quality declines.

RNA interference (RNA interference, RNAi) is emerging the transcribing of grown up recent years Gene silence in level.It is that a kind of double-stranded RNA (double-stranded RNA, dsRNA) molecule is at mRNA That closes corresponding sequence gene in level expresses or makes the process of its silence, the most sequence-specific transcribes rear base Because of reticent (post-transcriptional gene silencing, PTGS).Different organisms, including planting Thing, fruit bat, nematicide and the big multipotency of some mammals utilize RNA interference mechanism to resist virus and other foreign nucleus Sour invades people.It is pointed out to be probably a kind of evolution conservative of organism mechanism.It is likely to become specificity reticent simultaneously Endogenous gene and a revolutionary instrument of gene function analysis.

Speculate that the main process that RNAi occurs is roughly divided into three phases according to some experimental results: (1) initial period, DsRNA is at the intracellular siRNA uniformly being cut into 21-25nt size by Rnase III (such as Dicer).DsRNA can To be external source (such as viral RNA replicative intermediate or the dsRNA that is manually imported), it is also possible to be endogenous (as carefully The dsRNA that in born of the same parents, single stranded RNA is formed under the effect of rna dependent rna polymerase (RdRP)).The length of siRNA is Race is special, and 5 ' ends are phosphorylations, and 3 ' ends often highlight 2 nts.(2) amplification stage, siRNA and mRNA Targeting combines, and as primer, again forms dsRNA, dsRNA and be cut into again siRNA under RdRP effect, The siRNA being newly formed can enter again next round circulation and reach the purpose of amplification.(3) effective stage, siNRA and Endonuclease and all formation RISC (RNA induced silencing complex) of other albumen.RISC's Nucleic acid moiety plays the effect of targeting, and protein part then plays the effect of degraded mRNA.The biological significance of RNAi It is that it serves the existence monitoring and suppressing abnormal or external source hereditary material at cell, maintenance heredity itself The stability of material.

Become duplex structure to lead a certain sequential design of virus to be allowed to express into plant, the strengthening of RNA silence can be induced Natural RNA silence anti-virus ability in plant, this makes the acquisition of transgenic plant of high antiviral become May.Waterhouse etc. utilize marmor upsilon (PVY) protease gene segment structure IRS carrier to carry out Nicotiana tabacum L. Convert, it is thus achieved that resistant plant.Wang etc. utilize containing luteovirus (Barley yellow dwarf virus, BYDV) the rdrp gene segment of PAV strain is built into the carrier that can produce hairpin-type RNA structure, and by this carrier Lead and obtain strong resistant plant into Fructus Hordei Vulgaris and obtain stable heredity.Kalantids etc. are turning cucumber mosaic virus The reconstituted tobacco plant of (Cucumber mosaic virus, CMV) cDNA inverted repeat detects one complete The strain of full resistance, and confirm that tobacco virus resistance is the most relevant to the generation of siRNA.Virus inoculation simultaneously Convert plant surface and reveal responsive type, recovery-type (performance symptom but the blade that newly grows is asymptomatic), complete resistance 3 Plant phenotype.Although responsive type therein is sensitive to virus, but symptom is more light than comparison.These three phenotype except Illustrating that RNA silence signal can spread outside generation systematicness silence in plant, also explanation utilizes RNA silence means The resistance intensity size of resistant plant that obtains is the most not quite identical.

On the technique study of RNA silence antiviral, Tenllado etc. observes with direct injection and agriculture bacillus mediated Convert two ways to lead into after plant leaf blade cell virus dsRNA it was found that both of which successfully stops Tobacco etch virus (Tobacco etch virus, TEV), capsaicinoid ointment (Pepper mild mottle Virus, PMMoV) and the infection processs of alfalfa mosaic virus (Alfalfa mosaic virus, AMV).Simultaneously he Also the dsRNA being used for direct injection is diluted process find dsRNA induction plant virus resistance be dsRNA Dosage relies on, and this is consistent with the conclusion drawn on animal.And test result indicate that, i.e. of Pandolfini etc. Make to lead plumpox virus (Plum pox virus, the PPV) genomic fragment into Nicotiana tabacum L. and contain the intron sequence that can shear Row, may also detect that the existence converting segment siRNA in transformant, and it are anti-to show the system infecting PPV Property.

Summary of the invention

The technical problem to be solved is to provide a kind of dual anti-iris cymbidium mosaic virus and tooth orchid ring speckle The RNAi carrier of virus, after making this carrier infect conversion iris by Agrobacterium, can disturb sword-leaved cymbidium floral leaf simultaneously Virus and odontoglossum ringspot virus (ORSV) coat protein gene are expressed, and can produce iris cymbidium mosaic virus simultaneously And the resistance of odontoglossum ring spot virus (ORSV) (CymMV).

The dual anti-iris cymbidium mosaic virus of the present invention and the RNAi carrier of odontoglossum ring spot virus, its sequence is such as Shown in Seq ID No:1.

The building process of described RNAi carrier is: choose cymbidium mosaic virus and odontoglossum ring spot virus shell egg respectively Conserved sequence (Seq ID No:2, Seq ID No:3) in white gene (CP)；By chimeric for two parts and structure Build inverted repeat (Seq ID No:4), be inserted into intron two ends, be cloned into binary plant expression vector In pCAMBIA1301-220, obtain RNAi plant binary expression vector pRNAi-CP.

What the present invention built utilizes dual anti-iris cymbidium mosaic virus and the RNAi carrier of odontoglossum ring spot virus, choosing Take the conserved sequence in cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene (CP), utilize hpRNA Silencing strategies is possible not only to obtain more resistance transfer-gen plant, and owing to not expressing virus protein, can reduce The misgivings of people's safety of transgenic crops to carrying viral gene, make that germplasm innovation purposiveness is higher the most more to be pacified Complete effective；Design target sequence in high conserved region section, and only need the homologous sequence of 20bp that virus just can be caused reticent, It is avoided that the resistance mutant that virus produces because of the high mutation rate in non-conservative district；Simultaneously can also be by sword-leaved cymbidium floral leaf Virus and odontoglossum ringspot virus (ORSV) coat protein gene sequence are together in series and obtain cymbidium mosaic virus and tooth orchid ring Pinta two viral resistance of wide spectrum of poison.The present invention is applied to by the dual anti-two-strain of gene transformation technology selection-breeding Iris new varieties, can effectively preventing iris produce on cymbidium mosaic virus and odontoglossum ring spot virus, produce Huge economic benefit.

Detailed description of the invention

The embodiment of the present invention is as follows:

1, build cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene merges fragment

Primer is designed respectively according to cymbidium mosaic virus in ncbi database and odontoglossum ringspot virus (ORSV) coat protein gene (table 1), amplification length is 238bp and 380bp, is utilized respectively primer CyMVCP-SacIF/CyMVCP-R And ORSVCP-F/ORSVCP-KpnR, with the cDNA containing cymbidium mosaic virus and odontoglossum ring spot virus as mould Plate, carries out pcr amplification reaction, amplification employing 50 μ L systems: template 2.5 μ L, each 2.5 μ L(10 μm ol/L of primer), The dNTP Mixture4.0 each 2.5mmol/L of μ L(), 10 × PCR Buffer (Mg2+plus) 5.0 μ L, TaqE0.5 μ L, 33 μ L sterilizing ultra-pure water polishings.Amplification program is: 94 DEG C of denaturations 5min；(94 DEG C of degeneration 30s, 55 DEG C Annealing 30s, 72 DEG C extend 1min) 35 circulations；10min is extended after 72 DEG C.PCR primer is with 1.2% Agarose gel carries out electrophoresis detection, is purified removal primer and primer dimer with recovery purification kit, returns Receive target stripe.

Utilize two virus coat protein gene sequences of fusion DNA vaccine technology, at primer CyMVCP-SacIF With under ORSVCP-KpnR effect, with the product after recovery as template, carry out PCR fusion.Merge and use 50 μ L System: each 2.5 μ L of template, each 2.5 μ L(10 μm ol/L of primer), the dNTP Mixture4.0 each 2.5mmol/L of μ L(), 10 × PCR Buffer (Mg2+plus) 5.0 μ L, TaqE0.5 μ L, 30.5 μ L sterilizing ultra-pure water polishings.Merge bar Part is with above PCR amplification program.PCR primer carries out electrophoresis detection with 1.2% agarose gel, with reclaiming purification Test kit is purified removal primer and primer dimer, reclaims the target stripe of 618bp.

Table 1PCR expands the primer sequence

2, RNAi plant binary expression vector pRNAi-CP builds

With the fusion DNA vaccine product of two viral CP genetic fragments as template, respectively with primer CyMVCP-SacIF/ ORSVCP-KpnR and CyMVCP-XbaIF/ORSVCP-SmaR carries out PCR amplification, and reaction condition is: 94 DEG C 3 min；94 DEG C of 45s, 55 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations；72℃10min.In advance Meter expands the purpose fragment of about 600bp, introduces restriction enzyme site Sac I, Kpn respectively at fragment 5' end and 3' end I and Sam I, Xba I.

With expression vector plasmid pHellsgate12 (GenBankCS591107) as template, design primer I ntronF With IntronR amplification intron fragment (Intron), introduce Sac I, Kpn I and respectively at the two ends of this gene Xba I, BamH I restricted enzyme point of contact.PCR response parameter is: 94 DEG C of 3min；94 DEG C of 45s, 57.5 DEG C of 1min, 72 DEG C of 1.5min, 30 circulations；72℃10min.

The RNAi of restructuring is expressed unit and plant expression carrier plasmid pCAMBIA1301-220 respectively with Sac I He BamH I double digestion, by the carrier wire plasmid DNA reclaimed with Insert Fragment DNA by 1: 3(mass ratio) carry out Mixing, converts bacillus coli DH 5 alpha after being oriented connection with T4DNA ligase, builds plant and interferes expression to carry Body pRNAi-CP.Detected by PCR and enzyme action, identify the escherichia coli positive colony with RNAi carrier.

3, carrier conversion in Agrobacterium

The plant expression vector pRNAi-CP built is imported in Agrobacterium LBA4404 by freeze-thaw method, by profit good fortune Gentle kanamycin screens, it is thus achieved that resistant strain.The plasmid extracted from Agrobacterium extracts with from escherichia coli The band that goes out of plasmid amplification consistent, show that plasmid vector the most successfully proceeds in Agrobacterium LBA4404, can directly use In follow-up transgenic work.

4, the cultivation of vegetable material and conversion

4.1 Agrobacteriums infect the preparation of liquid

Picking carries the Agrobacterium LBA4404 list bacterium colony of plant Overexpression vector pRNAi-CP.It is inoculated in 5mL YEB(50mg/L Km+50mg/L Rif) in fluid medium, in 28 DEG C, 200rpm shaken overnight. Take 500 μ l bacterium solution next day and be transformed into YEB(50mg/L Km+50mg/L Rif fresh for 50mL) liquid training Supporting in base in 28 DEG C, 200rpm shaken overnight, is 0.5～0.6 to bacterium solution OD600.

4.2 iris Protocorms

The kind pod of results iris, after 75% alcohol disinfecting, uses dissecting knife strip off on superclean bench, Seed is seeded in KC+6-BA6.0mg/L+NAA0.2mg/L+AC0.5mg/L+ potato juice 100g/L culture medium On, under dark condition, 25 DEG C, cultivate and within 50 days, induce the tiny protocorm of iris, and in same culture medium Upper propagation.

The infecting and co-culture of 4.3 outer implant

Cultured bacterium solution is used 50mLKC+6-BA6.0mg/L+NAA0.2 after centrifugal 12 minutes under 4500rpm Fluid medium (adding AS to final concentration 100 μMs) the resuspended thalline of mg/L+ sucrose 20mg/L, then will solve Cut open the protocorm that pin pricked and move in ready bacterium solution, 28 DEG C, several minutes of vibrating under the conditions of 120rpm, take Go out protocorm to be placed on KC+6-BA6.0mg/L+NAA0.2mg/L+ sucrose 20mg/L solid medium, in After at 25 DEG C, light culture co-cultures for 2-6 days, protocorm tissue is proceeded in the empty triangular flask of sterilizing in advance, with aseptic Water rinses 3-5 time, then embraces the agriculture bar on mycin sterilized water (head containing 300mg/L embraces mycin) removing protocorm surface with head Bacterium, then transfer to micro-organisms after be placed on aseptic filter paper the moisture blotting surface attachment for 1 time with aseptic water washing Base carries out antibacterial screening.

4.3 adventitious shoot regenerations, screening and Plantlet formation

Iris protocorm after cleaning is seeded in KC+6-BA6.0mg/L+NAA0.2mg/L+ head and embraces mycin 20 KC+6-BA6.0mg/L+NAA0.2mg/L+ kanamycin is proceeded to after cultivating 30 days on the micro-organisms base of mg/L Resistance screening is carried out 1 month in the screening culture medium of 500mg/L.It is placed in KC+6-BA6.0mg/L+NAA0.2mg/L again One is screened under+AC0.5mg/L+ potato juice 150g/L+Km500mg/L+Cef200mg/L culture medium Month, the protocorm after selection is placed in the KC+3.0mg mg/L6-BA+NAA0.5mg/L+AC of additional 200mg/L Cef Carry out, on 0.5mg/L+ potato juice 150g/L division culture medium, cultivation of sprouting, transfer to add by the plant of differentiation There are 150mg/L kanamycin and the KC+6-BA2.0mg/L+NAA0.5mg/L+AC1mg/L of 200mg/L Cef + potato juice 150g/L root media carries out root culture.

Eventually pass root culture until producing complete regeneration plant, the plant corkage seedling exercising that choosing is grown fine, wash Going the culture medium of root, move in the nutritive cube equipped with compost (peat soil: Vermiculitum=3:1), plastic sheeting covers Lid moisturizing 2～press seedling plant normal management after 3 days.

5, the qualification of transfer-gen plant

5.1DNA extracts and PCR detection

Utilize the insertion of exogenous sequences in the obtained resistant plant genome of PCR method detection, identify transgenic with this Plant.DNA(CTAB method is extracted respectively) from the Phalaenopsis leaves of non-transgenic negative control and transgenic.Profit With two-strain CP gene fusion fragment amplification primer CyMVCP-SacIF/ORSVCP-KpnR, preliminary identification is turned The carrying out of gene plant detects further.PCR response procedures is: 94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C 1 Min, 72 DEG C of 1min, 32 circulations, 72 DEG C of 10min.The fragment amplifying about 2200bp shows hair clip Structure has been integrated in iris genome.

5.2 transgenic iris Disease Resistance Identifications

Infect cymbidium mosaic virus and the Phalaenopsis leaves of odontoglossum ring spot virus, juice from producing greenhouse is chosen simultaneously Liquid is seeded on iris transfer-gen plant and nontransgenic plants blade simultaneously.After 4 weeks, nontransgenic plants occurs The cymbidium mosaic viruses such as floral leaf and the classical symptom of odontoglossum ring spot virus, but transfer-gen plant does not has symptom.

Extract iris transfer-gen plant and the total serum IgE of nontransgenic plants blade respectively, after reverse transcription, use RT-PCR Method detection cymbidium mosaic virus and odontoglossum ring spot virus.Primer is respectively according to sword-leaved cymbidium floral leaf in ncbi database Virus and odontoglossum ring spot virus rdrp virus RdRp design, and amplification length is the fragment of 287bp and 387bp. Amplification system and PCR response procedures are ibid.Result shows, nontransgenic plants amplifiable go out the sword-leaved cymbidium floral leaf such as floral leaf Virus and the band of odontoglossum ring spot virus, but transfer-gen plant does not has.

These results indicate that after the carrier built infects conversion iris by Agrobacterium, Flos Cymbidii Ensifolii can be disturbed simultaneously Mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene are expressed, and can produce iris cymbidium mosaic virus simultaneously And the resistance of odontoglossum ring spot virus (ORSV) (CymMV).

Table 2 cymbidium mosaic virus and odontoglossum ring spot virus Viral diagnosis primer

Sequence table

<110>Zhengjiang City's Vegetable Research Institute

<120>dual anti-iris cymbidium mosaic virus and the RNAi carrier of odontoglossum ring spot virus

<130> 2014

<160> 4

<170> PatentIn version 3.3

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ttgtagtttt attaacttct aaatggattg actattaatt aaatgaatta gtcgaacatg 1320

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aatgtaaaaa aatatgacaa gtgatgtgta agacgaagaa gataaaagtt gagagtaagt 180

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aagctatgta atatttgcgc cataaccaat caattaaaaa atagatcagt ttaaagaaag 900

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Claims (3)

1. dual anti-iris cymbidium mosaic virus and a RNAi carrier for odontoglossum ring spot virus, its sequence such as Seq Shown in ID No:1.

Dual anti-iris cymbidium mosaic virus the most according to claim 1 and the RNAi of odontoglossum ring spot virus Carrier, is characterized in that: the building process of described RNAi carrier is, chooses cymbidium mosaic virus and tooth orchid ring respectively Conserved sequence Seq ID No:2 in speckle virus coat protein gene, Seq ID No:3；Two parts are fitted together to also Build inverted repeat Seq ID No:4, be inserted into intron two ends, be cloned into binary plant expression vector In pCAMBIA1301-220, obtain RNAi plant binary expression vector pRNAi-CP.

3. the RNAi of dual anti-iris cymbidium mosaic virus described in a claim 1 and odontoglossum ring spot virus carries Body is preventing and treating cymbidium mosaic virus and the application of odontoglossum ring spot virus that iris produces.