CN103993033A - RNAi vector with resistance to both cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) of Phalaenopsis ssp. - Google Patents
RNAi vector with resistance to both cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) of Phalaenopsis ssp. Download PDFInfo
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- CN103993033A CN103993033A CN201410049351.1A CN201410049351A CN103993033A CN 103993033 A CN103993033 A CN 103993033A CN 201410049351 A CN201410049351 A CN 201410049351A CN 103993033 A CN103993033 A CN 103993033A
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Abstract
The invention belongs to the field of molecular biology and relates to an RNAi vector with resistance to both CymMV and ORSV of Phalaenopsis ssp. and application of the RNAi vector in mediation of resistance to both CymMV and ORSV of Phalaenopsis ssp. The RNAi vector with resistance to both CymMV and ORSV of Phalaenopsis ssp. has a sequence as shown in Seq ID No. 1. The RNAi vector is applied to breeding of a novel variety of Phalaenopsis ssp. with resistance to both CymMV and ORSV by using gene transformation technology, can effectively prevent CymMV and ORSV in production of Phalaenopsis ssp. and enables immense economic benefits to be produced.
Description
Technical field
The invention belongs to biology field, the present invention relates to the RNAi carrier of a kind of dual anti-butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus, with and mediation to the application in the resistance of butterfly orchid cymbidium mosaic virus (CymMV) and odontoglossum ring spot virus (ORSV).
Background technology
Butterfly orchid (Phalaenopsis ssp.) is the orchid family Phalaenopsis flowers, in 1750, is found the earliest, has found so far more than 70 initial species, on TaiWan, China and Thailand, Philippines, Malaysia, Indonesia and other places, all has distribution.Butterfly orchid is because of its flower shape grace, color is magnificent, and the treasure in tropical orchid has the good reputation of " queen in orchid ", the new lover who add storage tolerance, be suitable for indoor placement, economic benefit advantages of higher becomes flower industry, butterfly orchid potted flower has become the important agricultural industry in the whole world.China's butterfly orchid production development is in recent years rapid, and within 1997, continent butterfly orchid manufacturing enterprise is very few, and year landings only have 200,000 strain left and right, and surpassing 1,000 ten thousand strain to butterfly orchid landings in 2003, the output value reaches 500,000,000 yuan, 2010, China's butterfly orchid annual flower landings reached 2,610 ten thousand strains.
Along with the implant mass of China's butterfly orchid, disease is also on the rise.Butterfly orchid virus disease is the important disease of a class on butterfly orchid, because butterfly orchid relies on tissue culture to carry out vegetative propagation mostly on producing, virus is constantly accumulation in vegetative organ, virus also can be propagated by the wound that in tissue culture and greenhouse, plant management causes simultaneously, make butterfly orchid prevention and cure of viruses difficulty, have a strong impact on growth and the ornamental quality of butterfly orchid, and the country such as American-European is very strict to the quarantine of butterfly orchid virus, with outlet butterfly orchid seedling and the finished product of virus disease, to Enterprises for Export in China, bring about great losses.On producing at present, also do not have effective medicine to prevent and treat butterfly orchid virus disease, can only prevent and treat by eliminating diseased plant and stem tip culture detoxification, but Shoot-tip Grafting In Vitro operation easier is large, expend a large amount of manpower and materials, detoxification efficiency is sometimes unsatisfactory, and in expanding numerous and cultivation process, also can infect virus, therefore utilize the technology seed selection butterfly orchid disease-resistant varieties such as genetically engineered become in production in the urgent need to.
At present, the orchid virus of having reported in the world has: cymbidium mosaic virus, odontoglossum ring spot virus, sword-leaved cymbidium mild mosaic poison, cybidium ring spot virus, annulus zonatus, about 10 kinds of viruses such as orchid mosaic virus, dendrobium mosaic virus, cattleya mottle virus and cucumber mosaic virus all ages, wherein with cymbidium mosaic virus (Cymbidium mosaic virus, CymMV) and odontoglossum ring spot virus (Odontoglossum ringspot virus, ORSV) occur the most general.Cymbidium mosaic virus and odontoglossum ring spot virus are two kinds of main virus causing diseases that infect butterfly orchid.
CymMV is subordinate to linear Viraceae, potexvirus, the 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 infect separately or Combined Infection orchid, cause the symptoms such as floral leaf, necrosis, vein chlorisis, streak, during Combined Infection, can cause plant dwarfing, the severes such as leaf malformation, cause the blue serious plant disease that produces of the multiple tropical Lan Ji state such as butterfly orchid, stone Mongolian oak orchid, great Hua favour orchid, oncidiumLuridum, greatly reduce the commodity value of orchid.China report orchid virus starts from nineteen eighty-three Zhu Benming etc. and is separated to two-strain from the orchid in Shanghai, is accredited as cymbidium mosaic virus and odontoglossum ring spot virus.Shen Shulin (1990) is to the biology of this two-strain, the further systematic study of biochemical characteristic, and confirmation is topmost virus on China orchid.
Butterfly orchid virus is mainly propagated by amboceptor or juice.Amboceptor is important route of transmission, can propagate by aphid, nematode, mite class etc.It is the phase mutual friction by blade that juice is propagated, or people's tissue culture and farm work, makes healthy leaves cause microtrauma, causes virus disseminating.By above-mentioned two kinds of approach, make butterfly orchid infect virus, and reach next asexual generation by tissue culture expanding propagation, thereby accumulation for a long time in butterfly orchid nourishing body causes yield and quality to decline.
It is the emerging Gene silence on transcriptional level growing up recent years that RNA disturbs (RNA interference, RNAi).A kind of double-stranded RNA (double-stranded RNA, dsRNA) molecule is closed the expression of corresponding sequence gene or is made its reticent process in mRNA level, sequence-specific PTGS (post-transcriptional gene silencing, PTGS) namely.Different organisms, comprises that plant, fruit bat, nematode and the large multipotency of some Mammalss utilize RNA interference mechanism to resist the people that invades of virus and other foreign nucleus acid.Pointing out it may be a kind of evolution conservative mechanism of organism.Also may become a revolutionary instrument of the reticent native gene of specificity and gene function analysis simultaneously.
According to some experimental results, infer that the main process that RNAi occurs is roughly divided into three phases: (1) initial period, dsRNA is evenly cut into the siRNA of 21-25nt size in cell by Rnase III (as Dicer).DsRNA can be external source (as viral RNA replicative intermediate or the artificial dsRNA importing), can be also endogenous (dsRNA forming under the effect of rna dependent rna polysaccharase (RdRP) as single stranded RNA in cell).The length of siRNA is that race is special, and 5 ' end is phosphorylation, and 3 ' end is outstanding 2 nts often.(2) the amplification stage, siRNA is combined with mRNA targeting, and as primer, again forms dsRNA under RdRP effect, and dsRNA is cut into again siRNA, and the new siRNA forming can enter again next round circulation and reach the object of amplification.(3) effective stage, all form RISC (RNA induced silencing complex) siNRA and endonuclease and other albumen.The nucleic acid moiety of RISC plays the effect of targeting, and protein part plays the effect of degraded mRNA.The biological significance of RNAi is just that it has played and monitors and suppress genetic material abnormal or external source in the existence of cell, maintains the stability of genetic material own.
Become duplex structure to lead into plant materials a certain sequences Design of virus and make it to express, can bring out the reticent anti-virus ability of natural RNA in the reticent fortification of plants body of RNA, this makes the acquisition of the transgenic plant of high antiviral become possibility.Waterhouse etc. utilize marmor upsilon (PVY) proteinase gene segment structure IRS carrier to carry out Transformation of tobacco, obtain resistant plant.The utilizations such as Wang contain barly yellow dwarf virus (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 this carrier is led into barley and obtained strong resistant plant and obtain genetic stability.Kalantids etc. detect the strain of a complete resistance in the reconstituted tobacco plant that turns cucumber mosaic virus (Cucumber mosaic virus, CMV) cDNA inverted repeats, and confirm that tobacco virus resistance is directly relevant to the generation of siRNA.Simultaneously the conversion of plant body surface of virus inoculation reveals responsive type, recovery type (reveal any symptoms but the blade that newly grows is asymptomatic), 3 kinds of phenotypes of complete resistance.Although responsive type is wherein responsive to virus, symptom is more light than contrast.These three kinds of phenotypes can spread the systemic silence of generation except the reticent signal of RNA is described in plant materials, also illustrate that the resistance intensity size of the resistant plant that utilizes the reticent means acquisition of RNA is also not quite identical.
In the reticent antiviral method research of RNA; Tenllado etc. have observed with direct injection and two kinds of modes of agrobacterium mediation converted viral dsRNA have been led into found that after plant leaf cell; two kinds of modes have all successfully stoped tobacco etch virus (Tobacco etch virus; TEV), the light mottle virus of capsicum (Pepper mild mottle virus; PMMoV) and the infection processs of alfalfa mosaic virus (Alfalfa mosaic virus, AMV).The plant virus resistance that they also carry out the dsRNA for direct injection dilution process discovery dsRNA induction is simultaneously that dsRNA dosage relies on, and this is consistent with the conclusion drawing on animal.And the experimental result of Pandolfini etc. shows, even if lead plumpox virus (the Plum pox virus into tobacco, PPV) genomic fragment contains the intron sequences that can shear, the existence that transforms segment siRNA in transformant, also can be detected, and show the system resistance that PPV is infected.
Summary of the invention
Technical problem to be solved by this invention is, the RNAi carrier of a kind of dual anti-butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus is provided, make this carrier infect and transform after butterfly orchid by Agrobacterium, can disturb cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene to express simultaneously, can produce the resistance to butterfly orchid cymbidium mosaic virus (CymMV) and odontoglossum ring spot virus (ORSV) simultaneously.
The RNAi carrier of dual anti-butterfly orchid cymbidium mosaic virus of the present invention and odontoglossum ring spot virus, its sequence is as shown in Seq ID No:1.
The building process of described RNAi carrier is: choose respectively the conserved sequence (Seq ID No:2, Seq ID No:3) in cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene (CP); Two portions are chimeric and build inverted repeats (Seq ID No:4), be inserted into intron two ends, be cloned in double base plant expression vector pCAMBIA1301-220, obtain RNAi plant binary expression vector pRNAi-CP.
The RNAi carrier that utilizes dual anti-butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus that the present invention builds, choose the conserved sequence in cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene (CP), utilize the reticent strategy of hpRNA not only can obtain the transfer-gen plant of higher resistance, and owing to not expressing viral protein, can reduce people to carrying the misgivings of the safety of transgenic crops of virogene, make germplasm innovation purpose stronger also more safe and effective; At high conserved region section design target sequence, and only need the homologous sequence of 20bp just can cause that virus is reticent, can avoid the viral resistance mutant strain producing because of the high mutation rate in non-conservative district; Cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene sequence can also be together in series and obtain cymbidium mosaic virus and two viral resistance of wide spectrum of odontoglossum ring spot virus simultaneously.The present invention is applied to by the butterfly orchid new variety of the dual anti-two-strain of gene transformation technology seed selection, and cymbidium mosaic virus and the odontoglossum ring spot virus can effectively preventing butterfly orchid produced, produce huge economic benefit.
Embodiment
The embodiment of the present invention is as follows:
1, build cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene and merge fragment
Respectively according to cymbidium mosaic virus in ncbi database and odontoglossum ringspot virus (ORSV) coat protein gene design primer (table 1), amplification length is 238bp and 380bp, utilize respectively primer CyMVCP-SacIF/CyMVCP-R and ORSVCP-F/ORSVCP-KpnR, the cDNA that contains cymbidium mosaic virus and odontoglossum ring spot virus of take is template, carry out pcr amplification reaction, amplification adopts 50 μ L systems: template 2.5 μ L, each 2.5 μ L(10 μ mol/L of primer), each 2.5mmol/L of dNTP Mixture4.0 μ L(), 10 * PCR Buffer (Mg2+plus), 5.0 μ L, TaqE0.5 μ L, 33 μ L sterilizing ultrapure water polishings.Amplification program is: 94 ℃ of denaturation 5min; (72 ℃ are extended 1min for 94 ℃ of sex change 30s, 55 ℃ of annealing 30s) 35 circulations; After 72 ℃, extend 10min.PCR product carries out electrophoresis detection with 1.2% sepharose, carries out purifying remove primer and primer dimer with recovery purification kit, reclaims target stripe.
Utilize and merge two virus coat protein gene sequences of round pcr connection, under primer CyMVCP-SacIF and ORSVCP-KpnR effect, the product of take after reclaiming is template, carries out PCR fusion.Merge the 50 μ L systems of employing: each 2.5 μ L of template, each 2.5 μ L(10 μ mol/L of primer), each 2.5mmol/L of dNTP Mixture4.0 μ L(), 10 * PCR Buffer (Mg2+plus), 5.0 μ L, TaqE0.5 μ L, 30.5 μ L sterilizing ultrapure water polishings.Fusion conditions is with above pcr amplification program.PCR product carries out electrophoresis detection with 1.2% sepharose, carries out purifying remove primer and primer dimer with recovery purification kit, reclaims the target stripe of 618bp.
Table 1PCR amplification the primer sequence
2, RNAi plant binary expression vector pRNAi-CP builds
The fusion PCR product of two viral CP gene fragments of take is template, carries out pcr amplification respectively with primer CyMVCP-SacIF/ORSVCP-KpnR and CyMVCP-XbaIF/ORSVCP-SmaR, and reaction conditions is: 94 ℃ of 3min; 94 ℃ of 45s, 55 ℃ of 1min, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.Estimate the object fragment of the about 600bp of amplification, at fragment 5' end and 3' end, introduce respectively restriction enzyme site Sac I, Kpn I and Sam I, Xba I.
The expression vector plasmid pHellsgate12 (GenBankCS591107) of take is template, design primer I ntronF and IntronR amplification intron fragment (Intron), introduce respectively Sac I, Kpn I and Xba I, BamH I restriction enzyme point of contact at the two ends of this gene.PCR reaction parameter is: 94 ℃ of 3min; 94 ℃ of 45s, 57.5 ℃ of 1min, 72 ℃ of 1.5min, 30 circulations; 72 ℃ of 10min.
RNAi expression unit and the plant expression carrier plasmid pCAMBIA1301-220 of restructuring are used respectively to Sac I and BamH I double digestion, by the carrier wire plasmid DNA reclaiming with Insert Fragment DNA by 1: 3(mass ratio) mix, with T4DNA ligase enzyme, carry out after orientation connects transforming bacillus coli DH 5 alpha, build plant and interfere expression vector pRNAi-CP.By PCR and enzyme, cut detection, identify the intestinal bacteria positive colony with RNAi carrier.
3, the conversion of carrier in Agrobacterium
The plant expression vector pRNAi-CP building is imported in Agrobacterium LBA4404 by freeze-thaw method, with Rifampin and kantlex, screen, obtain resistant strain.The plasmid extracting from Agrobacterium is consistent with the band that the plasmid amplification extracting from intestinal bacteria goes out, and shows that plasmid vector successfully proceeds in Agrobacterium LBA4404, can be directly used in follow-up transgenosis work.
4, the cultivation of vegetable material and conversion
4.1 Agrobacteriums are infected the preparation of liquid
Picking carries the Agrobacterium LBA4404 list bacterium colony of plant Overexpression vector pRNAi-CP.Be inoculated in 5mLYEB(50mg/L Km+50mg/L Rif) in liquid nutrient medium, in 28 ℃, 200rpm shaken overnight.Get 500 μ l bacterium liquid next day and be transformed into the YEB(50mg/L Km+50mg/L Rif that 50mL is fresh) in liquid nutrient medium in 28 ℃, 200rpm shaken overnight, to bacterium liquid OD600 be 0.5～0.6.
4.2 butterfly orchid Protocorms
The kind pod of results butterfly orchid, after 75% alcohol disinfecting, on Bechtop, use scalper strip off, seed is seeded on KC+6-BA6.0mg/L+NAA0.2mg/L+AC0.5mg/L+ potato juice 100g/L substratum, under dark condition, 25 ℃, cultivate and within 50 days, induce the tiny protocorm of butterfly orchid, and breed on same substratum.
Infecting and cultivation altogether of 4.3 explants
By cultured bacterium liquid under 4500rpm after centrifugal 12 minutes with the resuspended thalline of liquid nutrient medium (adding AS to final concentration 100 μ M) of 50mLKC+6-BA6.0mg/L+NAA0.2mg/L+ sucrose 20mg/L, then the protocorm of dissection pinprick being crossed moves in ready bacterium liquid, at 28 ℃, under 120rpm condition, vibrate several minutes, taking out protocorm is placed on KC+6-BA6.0mg/L+NAA0.2mg/L+ sucrose 20mg/L solid medium, after at 25 ℃, dark cultivation is cultivated for 2-6 days altogether, protocorm tissue is proceeded in the empty triangular flask of sterilizing in advance, with aseptic water washing 3-5 time, with head, embrace the Agrobacterium that mycin sterilized water (embracing mycin containing 300mg/L head) is removed protocorm surface again, with aseptic water washing, be placed on for 1 time and transfer to micro-organisms base after the moisture that blots surface attachment on aseptic filter paper and carry out antibacterial screening again.
4.3 adventitious shoot regenerations, screening and Plantlet formation
Butterfly orchid protocorm after cleaning is seeded in to KC+6-BA6.0mg/L+NAA0.2mg/L+ head to be embraced on the micro-organisms base of mycin 20mg/L, to cultivate in the screening culture medium that proceeds to KC+6-BA6.0mg/L+NAA0.2mg/L+ kantlex 500mg/L after 30 days and carries out resistance screening 1 month.Be placed in again under KC+6-BA6.0mg/L+NAA0.2mg/L+AC0.5mg/L+ potato juice 150g/L+Km500mg/L+Cef200mg/L substratum and screen one month, protocorm after selection is placed in the cultivation of sprouting on the KC+3.0mg mg/L6-BA+NAA0.5mg/L+AC0.5mg/L+ potato juice 150g/L division culture medium of additional 200mg/L Cef, and the plant of differentiation is transferred in the KC+6-BA2.0mg/L+NAA0.5mg/L+AC1mg/L+ potato juice 150g/L root media that is added with 150mg/L kantlex and 200mg/L Cef and carries out root culture.
Finally by crossing root culture until produce complete regeneration plant, the plant uncork hardening that choosing is grown fine, wash away the substratum of root, move to compost (peat soil: in nutrition pot vermiculite=3:1), plastics film covers moisturizing and presses seedling plant normal management after 2～3 days is housed.
5, the evaluation of transfer-gen plant
5.1DNA extracts and PCR detects
Utilize PCR method to detect the insertion of external source fragment in resulting resistant plant genome, with this, identify transfer-gen plant.From non-transgenic negative control and genetically modified Phalaenopsis leaves, extract DNA(CTAB method respectively).Utilize two-strain CP gene fusion fragment amplification primer CyMVCP-SacIF/ORSVCP-KpnR, the carrying out of the transfer-gen plant of preliminary identification further detected.PCR response procedures is: 94 ℃ of 5min, 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, 32 circulations, 72 ℃ of 10min.The fragment that amplifies 2200bp left and right shows that hairpin structure has been incorporated in butterfly orchid genome.
5.2 transgenosis butterfly orchid Disease Resistance Identifications
From production greenhouse, choose the Phalaenopsis leaves that simultaneously infects cymbidium mosaic virus and odontoglossum ring spot virus, juice is seeded on butterfly orchid transfer-gen plant and non-transgenic plant leaf simultaneously.After 4 weeks there is the classical symptom of the cymbidium mosaic viruses such as floral leaf and odontoglossum ring spot virus in non-transgenic plant, but transfer-gen plant does not have symptom.
Extract respectively total RNA of butterfly orchid transfer-gen plant and non-transgenic plant leaf, the method with RT-PCR after reverse transcription detects cymbidium mosaic virus and odontoglossum ring spot virus.Primer is respectively according to cymbidium mosaic virus in ncbi database 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 the same.Result shows, non-transgenic plant can amplify the band of the cymbidium mosaic viruses such as floral leaf and odontoglossum ring spot virus, but transfer-gen plant does not have.
These results show, the carrier building is infected and is transformed after butterfly orchid by Agrobacterium, can disturb cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene to express simultaneously, can produce the resistance to butterfly orchid cymbidium mosaic virus (CymMV) and odontoglossum ring spot virus (ORSV) simultaneously.
Table 2 cymbidium mosaic virus and odontoglossum ring spot virus virus detect primer
Claims (3)
1. a RNAi carrier for dual anti-butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus, its sequence is as shown in Seq ID No:1.
2. the RNAi carrier of dual anti-butterfly orchid cymbidium mosaic virus according to claim 1 and odontoglossum ring spot virus, it is characterized in that: the building process of described RNAi carrier is to choose respectively conserved sequence Seq ID No:2, Seq ID No:3 in cymbidium mosaic virus and odontoglossum ringspot virus (ORSV) coat protein gene; Two portions are chimeric and build inverted repeats Seq ID No:4, be inserted into intron two ends, be cloned in double base plant expression vector pCAMBIA1301-220, obtain RNAi plant binary expression vector pRNAi-CP.
3. described in claim 1, the RNAi carrier of dual anti-butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus is produced for preventing and treating butterfly orchid cymbidium mosaic virus and odontoglossum ring spot virus.
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| CN101781656A (en) * | 2009-10-19 | 2010-07-21 | 浙江大学 | Expression of odontoglossum ringspot virus (ORSV) coat protein gene and preparation method of antibody |
| TW201035320A (en) * | 2009-03-20 | 2010-10-01 | Taiwan Seed Improvement And Propagation Station Coa R O C | Quick testing primer set for outer root sheath protein and RdRp (RNA-directed RNA polymerase) of Orchid CymMV and ORSV viruses |
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