CN110511955A - Plant cytoplasm rhabdovirus infectious clone, expression vector and its construction method and application - Google Patents
Plant cytoplasm rhabdovirus infectious clone, expression vector and its construction method and application Download PDFInfo
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Abstract
The invention belongs to gene engineering technology field, a kind of plant cytoplasm rhabdovirus infectious clone, expression vector and its construction method and application are specifically disclosed.Plant cytoplasm Rhabdovirus expression vectors provided by the present invention are the carrier containing barley Huang item point mosaic virus (BYSMV) full-length genome reverse complementary strand.The present invention provides a kind of strategy of building worm biography plant virus infectious clone for the first time, i.e. by the way that the carrier of the viral components such as the nucleocapsid (N) of foregoing expression vectors and expression BYSMV, phosphoprotein (P), big polymerase protein (L) is established infectivity virus in this life cigarette through mediated by agriculture bacillus, recycles this life cigarette morbidity juice to be inoculated with monocotyledon by plant hopper insect and establish systematicness and infect.Moreover, the present invention still further provide the plant cytoplasm Rhabdovirus expression vectors carried out in monocotyledon or plant hopper insect bodies albumen high efficient expression or in terms of extensive use.
Description
Technical field
The invention belongs to gene engineering technology fields, specifically, be related to plant cytoplasm rhabdovirus infectious clone,
Expression vector and its construction method and application.
Background technique
There are the advantages such as synthesis, folding and posttranslational modification using plant in plant genetic engineering, it can be low with plant
At local mass production foreign protein, such as vaccine antigen, antibody and pharmaceutical protein etc..Most common method is to utilize to turn base
Because technology by the gene integration of foreign protein into Plant Genome, can continuously express foreign protein.This stabilization
The method expression quantity of conversion is generally lower, and transgenic protocol is more time-consuming, and there are also some monocot crops heredity to turn
Change extremely difficult, these factors have seriously affected the industrialization process using genetically modified plants as bioreactor.
Since the eighties in last century, scientist starts to explore using plant viral vector efficiently expressing exogenous gene,
Basic process is to carry out genetic manipulation to its viral genome on the basis of plant virus infectious clone, can be by purpose
Channel genes are into viral vectors, mass expressing external albumen when being proliferated with virus infection host cell.It is steady with gene genetic
Fixed conversion is compared, and viral vectors importing process is simple, does not need that artificially directly target gene is inserted on plant chromosome, because
This is short from the period for being building up to expression；Further, since a large amount of proliferation of the virus in host cell, the foreign protein of expression is also therewith
Increase, it is general hundreds times higher than stablizing transgenosis.The virus expression carrier constructed at present is in exogenous protein expression, functional genome
Research and other field application all play an important role.
Currently, plant virus-based expression vector is mainly with positive chain RNA virus using relatively broad, for example, Cauliflower Mosaic
Viral (CaMV), potato virus X (Potato virus X, PVX), Tobacco rattle virus (Tobacco rattle virus,
TRV), tobacco mosaic virus (TMV) (Tobacco mosaic virus, TMV), cucumber mosaic virus (Cucumber mosaic
Virus, CMV), Chinese pennisetum mosaic virus (Foxtail mosaic virus, FoMV), cowpea mosaic virus (Cowpea
Mosaic virus, CPMV), bromovirus (Brome mosaic virus, BMV).However, with above-mentioned positive chain RNA disease
The stability of virus expression carrier based on poison is poor, and the foreign gene being inserted into virus expression carrier is generally less than 500bp,
No longer than 1000bp, once being more than this range, exogenous sequences are easily lost.The inoculation method of virus expression carrier is generally logical
Agroinfiltration technology is crossed, and there is preferable effect generally on dicotyledon, is difficult on most of monocotyledons
Utilize agroinfiltration technology.
Moreover, the expression vector in insect is all using rhabdovirus expression vector, mainly in Lepidoptera at present
Foreign protein is expressed in insect or its cell line.But for the insect of Semiptera, having or not can express at present in adult
The effective expression carrier of foreign protein seriously hinders the progress and scientific prevention and cure of hemipteran.
Plant rhabdovirus is minus-stranded rna virus, 2015, sonchus yellow net rhabdovirus (Sonchus yellow
Net rhabdovirus, SYNV) infectious clone be reported first, be the infectious clone of first plant negative strand viruses,
Also for the building of other negative strand viruses provide reference (Jackson and Li, 2016；Wang et al., 2015).However, bitter
Lettuce dish Huang net rhabdovirus (SYNV) mainly infects dicotyledon by aphis propagation, cannot infect monocotyledon and fly
Lice class insect, therefore cannot function as the expression vector of monocot crops and plant hopper agricultural insect.
Therefore, monocotyledon and plant hopper insect can be infected by needing to study and develop one kind, and can express large fragment
The plant virus-based expression vector of foreign protein overcomes current plant virus-based expression vector in the limitation of application aspect.
Summary of the invention
In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of plant cytoplasm rhabdoviruses
Infectious clone, expression vector and its construction method and application.
In order to achieve the object of the present invention, technical scheme is as follows:
In a first aspect, providing a kind of plant cytoplasm rhabdovirus expression the present invention is based on plant cytoplasm rhabdovirus
Carrier, it is that and can express 2-3 foreign gene simultaneously within 5000bp which, which can stablize expression length,.
Moreover, the expression vector can in plant and plant hopper insect high efficient expression foreign protein, and be different from existing
There is the plant virus-based expression vector in technology, plant virus-based expression vector provided by the present invention being capable of the plant of systemic infection unifacial leaf
Object realizes high efficient expression of the foreign protein in monocotyledon.
Further, the plant cytoplasm rhabdovirus is barley Huang item point mosaic virus (BYSMV).
Specifically, plant cytoplasm Rhabdovirus expression vectors provided by the present invention, to contain BYSMV full-length genome
The carrier of reverse complementary strand.
BYSMV belongs to cytoplasm rhabdovirus, and genome is sub-thread strand RNA, overall length 12706nt, NCBI accession number
For KM213865.BYSMV genome can transcribe 9 mRNA, wherein a mRNA encodes two albumen, therefore BYSMV is compiled altogether
10 albumen of code, including 5 structural proteins and 5 auxilins.5 structural proteins include nucleocapsid protein
(nucleoprotein, N), phosphoprotein (phosphoprotein, P), stromatin (matrix protein, M), glycoprotein
(glycoprotein, G), big polymerase protein (large polymerase protein, L) (Yan et al., 2015).In
Between P and M albumen, four auxilins are encoded, are successively the encoder block overlapping of P3, P4/P5, P6, wherein P4 and P5；In G and L
Between albumen, P9 auxilin is encoded.
Since the barley Huang item point mosaic virus is minus-stranded rna virus, the reverse complementary strand of full-length genome is
For normal chain cDNA overall length.
Further, the present invention provides the construction method of foregoing expression vectors, the core contents of the construction method are as follows:
Reverse transcription and PCR amplification are carried out to the geneome RNA of barley Huang item point mosaic virus, normal chain cDNA is obtained, by the normal chain
CDNA is building up in carrier.
The carrier includes but is not limited to the carriers such as pXT1 or pCass4-Rz.Start containing cauliflower mosaic virus 35S
Son, Hepatitis C virus nucleic acid enzyme (HDVRz) sequence (NCBI accession number is L35896.1 or L35897.1) and transcription terminator sequences
The plant expression vector of (such as nopaline synthase terminator, NCBI accession number are AJ007624.1) can be used as the carrier into
The building of row plant cytoplasm Rhabdovirus expression vectors.
PXT1 carrier, complete sequence NCBI accession number are JN029690.1；PCass4-Rz carrier, can refer to and published
Document (P.Annamalai, A.L.N.Rao, Virology, 338 (2005) 96-11).In a specific embodiment of the invention
In, using carrier pXT1 as exemplary illustration.
Further, in the construction method, the mode of digestion connection or homologous recombination can be used by the normal chain
CDNA is building up in carrier.
Present invention warp experimental studies have found that, if BYSMV overall length is completely directly implemented into using routine experiment means
There is 1-4 pivotal nucleotide that mispairing occurs in pXT1, in BYSMV, infectious clone is caused to fail.In order to more preferably guarantee acquisition
The operability of the fidelity of BYSMV cDNA and final carrier, virus sequence can be divided into two sections or three sections and expand and be connected into
Carrier can effectively reduce error rate when construction cDNA clone.Second aspect, the present invention provides a kind of infectious clone, institutes
It states infectious clone carrier as described in (1) below mediated by agriculture bacillus~(2) or (1)~(3) and infects gained after this life cigarette altogether:
(1) afore-mentioned plants cytoplasm Rhabdovirus expression vectors；
(2) carrier of N, P, L of BYSMV are expressed respectively；Or the carrier of N, P, L of BYSMV are expressed simultaneously；
(3) simultaneously expressing gene silencing suppressor p19, HCpro and γ b carrier.
The present invention it has been investigated that, in (2), at the same express BYSMV N, P, L carrier infectivity effect much distinguish table
Up to the carrier of N, P, L.When (3) described carrier participation is infected altogether, the expression quantity of the described two carriers in (1)~(2) can be improved,
Greatly increase the success rate of infectious clone.
On this basis, the present invention provides the infectious clone new methods that a kind of worm passes plant virus, i.e., using infecting
This life cigarette transient expression is established cellular level and is infected, and gained infectivity virus injection mediator insect is inoculated with using mediator insect
And systemic infection monocotyledon.
The mediator insect is preferably plant hopper insect, including small brown rice planthopper, white backed planthopper and brown paddy plant hopper, but is not limited to
This.
The process after this life Tobacco Leaves infected addition viral extract specifically, by grinding, by what is obtained after grinding
Viral juice, or the viral crude extract obtained after the viral juice is further centrifuged, are injected in plant hopper insect bodies.
Further, the present invention provides the infectious clone in a kind of monocotyledon, by infecting institute after this life cigarette
The infectivity virus obtained, as infective vectors, infects gained after monocotyledon through plant hopper.
Wherein, the monocotyledon includes but is not limited to barley, wheat, millet, corn etc..
The third aspect, the present invention provides foregoing expression vectors answering in terms of infecting monocotyledon or plant hopper insect
With.
Further, based on the advantage of foregoing expression vectors, list is infected using the expression vector foreign gene-carrying
Cotyledon plant or plant hopper insect and expression alien gene, the foreign gene are length 5000bp, and the foreign gene is 1-3
A, i.e., the described expression vector can also express 2-3 foreign gene simultaneously.Expression vector of the present invention is relative to existing expression
There is especially apparent advantage in the expression to the large fragment foreign gene that length is 1000bp~5000bp in carrier.
When introducing foreign gene, preferably by after the foreign gene and the expression vector linearization for enzyme restriction, by same
The mode of source recombination is inserted between the N (nucleocapsid protein) of expression vector and P (phosphoprotein) or P (phosphoprotein) and P3 (Teng
Yan,et al.,2015,Virology,478: 112-122.)。
It should be noted that the position of insertion foreign gene is not limited to above-mentioned two position, in P6 and M (matrix egg
It is white), M (stromatin) and G (glycoprotein) can also design insertion position between G (glycoprotein) and P9 or P9 and L (replicase)
Point carries out exogenous protein expression.
Wherein, the restriction enzyme site of design may be selected the restriction enzyme site not having on overall length viral vectors, such as MluI,
BmtI or PacI etc., however, it is not limited to this.
More specifically, in above-mentioned preferred embodiment, two expression cassettes can be added between N and P, be separately added into MluI
And BmtI, the modes such as can connect after linearization for enzyme restriction by homologous recombination or digestion and be inserted into exogenous genetic fragment: P and P3 it
Between be inserted into PacI, exogenous genetic fragment can be inserted by way of homologous recombination after linearization for enzyme restriction.
Further, the expression characteristic of the plant cytoplasm rhabdovirus according to the present invention, just chain direction, close to 5 '
The gene expression amount at end is high, so if foreign gene needs high expression, is preferably inserted into first expression cassette after N；Work as external source
When gene needs low expression level, it is preferably inserted between P and P3.
Based on inventive concept of the invention, expression vector of the present invention also acts as the scientific research of protein function,
I.e. by overexpressing/being overexpressed the albumen of monocotyledon unknown function using the expression vector, infected list is observed
The phenotype of cotyledon plant, to analyze/identify the function of the albumen.
Therefore, the application the present invention also provides foregoing expression vectors in terms of Rapid identification gene function.
Fourth aspect, the present invention provides foregoing expression vectors to carry out gene volume to plant hopper insect or monocotyledon
Application in terms of volume can express Cas9 albumen and gRNA, or expression other types gene using the expression vector simultaneously
Element is edited, directly host plant genome or insect mediator genome are effectively edited.
The advantage to plant hopper insect or monocotyledonous gene editing is realized using expression vector of the present invention
It is, Cas9 by transgenosis mode without being integrated on plant hopper insect or monocotyledonous genome, only by instantaneous
The editor to its genome can be realized in expression, avoids the security risk of transgenosis.
In other words, expression vector of the present invention can be used as the means of delivery of gene editing, in plant (especially unifacial leaf
Plant) and/or insect in carry out gene editing application.
The present invention relates to raw material or reagent be ordinary commercial products, the operation being related to is unless otherwise specified
This field routine operation.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can be combined with each other, obtain specific embodiment party
Formula.
Beneficial effects of the present invention at least that:
The present invention makes public for the first time a kind of expression vector based on plant cytoplasm rhabdovirus in worldwide.
The expression vector at least has the advantage that
(1) foreign gene of expression large fragment can be stablized, and can be with systemic infection monocotyledon；
(2) relatively high in plant interior expression amount, the period is shorter；
(3) by plant hopper insect Vector transmission, and band is malicious all the life for insect, can be produced with a large amount of plant hosts of high-efficiency inoculating
The foreign protein of raw high abundance；
(4) host plant and plant hopper insect are only infected, does not infect other animals and people, it is highly-safe；
(5) multiple albumen can be expressed simultaneously, can be used for expressing several functional protein protomers, directly carried out internal
Assembling；
(6) gene expression of moment is only mediated, unconformity, will not be possible as genetically modified plants into host cell gene group
The biological safety of generation and public the problems such as receiving at heart.
(7) there are the advantages such as the translation modification in eukaryocyte.
The present invention is based further on the characteristics of expression vector and advantage, provides the expression vector in Rapid identification
Application and the plant cytoplasm viral vectors in terms of gene function is as editor's means of delivery, in plant and/or insect
The middle application for carrying out gene editing.
Detailed description of the invention
Fig. 1 is the building flow chart of pCB-BYSMV plasmid.
Fig. 2 is the building flow chart of pGD-VSRs plasmid and pGD-NPL plasmid；Wherein, A:pGD-VSRs plasmid, B:
PGD-NPL plasmid.
Fig. 3 is the building flow chart of the sub- expression vector pBYMR of micro-replication.
Fig. 4 observes to establish the fluorecyte of BYSMV micro-replication subsystem on this life cigarette, and Bars=100 μm.
Fig. 5 is pCB-BYSMV-RFP carrier schematic diagram.
Fig. 6 is that this life cigarette observes result；Wherein, A:pCB-BYSMV-RFP injects the single-cell fluorescence figure of this life cigarette, B: this
The BYSMV virion observed in raw cigarette sample.
Fig. 7 is the Fluirescence observation figure that pCB-BYSMV-RFP infects plant hopper；Left hand view flies for what pCB-BYSMV-RFP infected
Red fluorescence can be observed under body formula fluorescence microscope in lice；Right part of flg is healthy plant hopper (control).
Fig. 8 is the Fluirescence observation result that pCB-BYSMV-RFP infects inoculation barley after plant hopper.
Fig. 9 is pCB-BYSMV-EGFP expression vector skeleton.
Whole strain shows green fluorescence after Figure 10 infects barley for pCB-BYSMV-EGFP under long-wave ultra violet lamp.
Figure 11 can produce β-D- glucuronic acid enzyme-to-substrate through virus replication after injecting this life cigarette for pCB-BYSMV-GUS
Cell is blue after reaction, does not inject pGD-NPL as control.
Figure 12 is that pCB-BYSMV-GUS infects GUS coloration result after barley, and healthy barley is as a control group.
Figure 13 is pCB-BYSMV-EGFP-RFP dual-expression vector skeleton.
Figure 14 is that can observe GFP and RFP fluorescence simultaneously after pCB-BYSMV-EGFP-RFP infects plant hopper, and health flies
Lice is as control, Bars=1mm.
Figure 15 is that pCB-BYSMV-EGFP-RFP infects after barley can be same intracellular while observing GFP and RFP
Fluorescence.
Figure 16 is pCB-BYSMV-EGFP-RFP-ECFP multiple representation vector construction process.
Figure 17 is to detect CAS9 albumen table after BYSMV gene editing carrier (BYSMV-CAS9-16C) injects this life of 16c cigarette
It reaches.
Figure 18 is that this life of 16c cigarette injection BYSMV is edited carrier (BYSMV-CAS9-16C), extracts DNA after NdeI digestion,
PCR amplification detects specific band.
Specific embodiment
It will be appreciated that the following examples are given for illustrative purposes only, it is not used to the present invention
Range limited.Those skilled in the art without departing from the spirit and purpose of the present invention, can be to this hair
It is bright to carry out various modifications and replace.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The list of primers used in following embodiment
Embodiment 1
1, BYSMV full length cDNA clone constructs
A. reverse transcription-amplification obtains BYSMV full length cDNA sequence: taking the barley disease leaf of BYSMV systemic infection, utilizes
Trizol (Invitrogen) method extracts blade total serum IgE, with 3 ' terminal specific primer primer1 of viral genome, reverse transcriptase
(SuperScriptTMIII Reverse Transcriptase, Invitrogen) reverse transcription is carried out, it is complete to obtain viral genome
Long cDNA.
B. the front half section carrier pCB-BYSMV of BYSMV normal chain cDNA is obtained1-6131: primer primer1, primer2 are utilized,
Phusion high fidelity enzyme (Phusion High-Fidelity DNA Polymerase, NEB), with BYSMV obtained above
CDNA is the segment of template amplification BYSMV 1-6131bp, and agarose gel electrophoresis recycles after specific fragment with seamless cloning kit
The pXT1 that box (NEBuilder HiFi DNA Assembly Master Mix) recombination to StuI, SalI double digestion linearizes is carried
Body (GenBank:JN029690.1, Agricultural University Of Nanjing professor Tao little Rong present), obtains pCB-BYSMV by testing above1 -6131。
C. it obtains the overall length carrier pCB-BYSMV of BYSMV normal chain cDNA: utilizing primer primer3, primer4,
Phusion high fidelity enzyme, using BYSMV cDNA as template amplification BYSMV 6132-12706 segment.Ago-Gel recycling is special
The pCB-BYSMV linearized to SalI, SmaI double digestion is recombinated with seamless Cloning Kit after segment1-6131Plasmid.By above-mentioned
Step obtains pCB-BYSMV full length cDNA clone, transcribes in plant through 35S promoter and generates viral normal chain genome, and
And the end RNA3 ' carries HDVRz ribozyme sequence and then guarantees accurate generation viral RNA.In addition in the latter half 6131- of normal chain
It is inserted into SalI restriction enzyme site behind 6232nt, genetic manipulation is carried out to virus convenient for subsequent, building process is such as shown in (Fig. 1).
2, BYSMV micro-replication subsystem is established
A. take advantage of a situation expression vector pGD-N, pGD-P, pGD-L of N, P and L albumen of BYSMV are constructed: being obtained with step 1
PCB-BYSMV plasmid is amplification template, is utilized respectively primer primer5, primer6 amplification N protein sequence, primer
Primer7, primer8 expand P protein sequence, and primer9, primer10 expand L protein sequence, then using seamless connection examination
Agent box is recombinated respectively to the pGD carrier linearized through HindIII, is seen and is published document (Goodin et al., The
Plant Journal (2002) 31 (3), 375-383), thus obtain plasmid pGD-N, pGD-P, pGD-L.
B. construct while expressing the carrier pGD-VSRs of p19, HCpro and γ b:
Step 1: the building of the carriers such as pGD-p19, pGD-HCpro and pGD- γ b
PGD-p19 building, according to having reported the artificial synthesized base of p19 sequence (GenBank:AJ288942.1) in NCBI
Because of segment, and at segment both ends plus XhoI and SalI restriction enzyme site, recycling segment makes after XhoI and SalI are double digested
PGD carrier, which is connected to, with T4DNA ligase obtains carrier pGD-p19；
PGD-HCpro building, according to having reported that HCpro sequence (GenBank:KX832617.1) is artificial synthesized in NCBI
The genetic fragment and XhoI and SalI restriction enzyme site are added respectively at both ends, recycle segment after XhoI and SalI are double digested
PGD carrier, which is connected to, using T4DNA ligase obtains carrier pGD-HCpro；
PGD- γ b building, according to having reported the artificial synthesized gene of γ b sequence (GenBank:U13917.1) in NCBI
It segment and is added respectively at both ends plus XhoI and SalI restriction enzyme site, recycle segment makes after XhoI and SalI are double digested
PGD carrier, which is connected to, with T4DNA ligase obtains carrier pGD- γ b.
Step 2: the expression cassette of three repressors is connected into a carrier
35S promoter-p19-NOS is expanded by template primer primer11, primer12 of pGD-p19
Terminator sequence；35S promoter- is expanded by template primer primer13, primer14 of pGD-HCpro
HCpro-NOS terminator sequence；It is template primer15, primer16 as primer using pGD- γ b, expands 35S
Promoter- γ b-NOS terminator sequence；Finally expand by template primer primer17, primer18 of carrier pGD
Increase linearized vector.Carrier is blended in one with 4 sequences expanded above with reference to NEB seamless connection kit specification
In a reaction system, finally obtain plasmid pGD-VSRs, can in plant simultaneously express three RNA silencing suppressor p19,
HCpro and γ b, building process are as shown in Figure 2 A.
C. the sub- expression vector pBYMR of micro-replication is constructed: first with primer primer19, primer20 to plasmid mould
Plate pCB-BYSMV obtains retaining 3 ' UTR-trailer sequence plasmid of leader-N-P-L from connection after carrying out inverse PCR
pBYSMV-NP；Then with primer primer21, primer22 reversely expand linearized vector that pBYSMV-NP is obtained with through drawing
The GFP sequence recombination that object primer23, primer24 are expanded, and then obtain GFP reporter gene replacement N protein sequence
Plasmid pBYMR-GFP-P；Finally using primer primer19, primer25 linearized vector pBYMR-GFP-P and with through primer
The RFP sequence of primer26, primer27 amplification recombinates to obtain the micro-replication sublist of RFP reporter gene replacement P protein sequence
Up to carrier pBYMR, it is as shown in Figure 3 to construct process.
D. BYSMV micro-replication subsystem is established on this life cigarette: by plasmid pGD-N, pGD-P, pGD- achieved above
L, pGD-VSRs, pBYMR convert Agrobacterium competent cell EHA105 respectively, after taking 28 DEG C of positive colony to shake bacterium, are suspended in agriculture
Buffer (10mM 2-morpholine ethane sulfonic acid, 10mM MgCl is resuspended in bacillus2, 150 μM of acetosyringones).Utilize visible light light-splitting
Luminosity measure OD600 be resuspended bacterium concentration and according to following final concentration pGD-N (0.1OD600, similarly hereinafter), pGD-P (0.1),
By the asepsis injector of removal syringe needle by bacterium solution after pGD-L (0.3), pGD-VSRs (0.1), pBYMR (0.3) mixed bacteria liquid
It is injected into this life Tobacco Leaves for cultivating three weeks or so.Take injection blade under fluorescence microscope it can be observed that GFP after six days
With RFP fluorescent protein expression, and be in unicellular distribution, show the duplication that micro-replication subsystem is successfully established, and is cloned
Related elements can all function, and fluorescence results are as shown in Figure 4.
3, BYSMV overall length infectious clone constructs
A. pGD-NPL transient expression vector is constructed: to improve bacillus Expression efficiency, by pGD-N, pGD-P and pGD-L
In expression unit be connected in series in a transient expression vector, the method utilized is with the part B in step 2, by three expression cassettes
Segment and pGD carrier segments carry out homologous recombination, finally obtain plasmid pGD-NPL, and specific building process is as shown in Figure 2 B.It should
After plasmid converts Agrobacterium competence EHA105, contain pBYMR (OD 0.3) so that final concentration 0D,600 0.3 is same, pGD-VSRs
This life Tobacco Leaves are injected after the Agrobacterium mixing of (OD 0.1), successful expression in pBYMR is observed after six days, shows pGD-NPL
It constructs successfully, functional N, P and L albumen can be expressed simultaneously.
B. building carries the overall length infectious clone pCB-BYSMV-RFP of reporter gene RFP: for convenient for detection building
BYSMV cDNA clone it is whether effective, insert RFP reporter gene expression frame, structure between two genes of N and P of BYSMV
PCB-BYSMV-RFP plasmid is built.First with primer primer28, primer29 using pCB-BYSMV as template amplification
Then it is connected into pMD-19T carrier and obtains plasmid by leader upstream vector 1057bp to P protein gene 6 05bp segment
pBYSNP-T；Then using primer primer30, primer31 using pBYMR as template amplification N3U-RFP-P5UBetween sequence
The carrier pBYSNP-T that recombination is extremely linearized through primer primer32, primer33, and then obtain plasmid pBYSNP-RFP-T；Most
Pass through nothing from BYSMV sequence of the plasmid pBYSNP-RFP-T amplification containing RFP with primer primer28, primer29 again afterwards
Seam Cloning Kit, which is recombinated to the carrier pCB-BYSMV linearized through NotI, ClaI, obtains recombinant vector pCB-BYSMV-RFP,
Vector construction process is as shown in Figure 5.The plasmid, which goes out viral RNA in plant host vivo transcription, to transcribe generation by rdrp virus
One independent RFP mRNA can judge whether the cDNA clone of BYSMV has by fluorescence microscope RFP reporter gene
Effect.
Infectivity of the C.BYSMV overall length infectious clone pCB-BYSMV-RFP on this life cigarette: by plasmid obtained above
PCB-BYSMV-RFP converts Agrobacterium competence EHA105, with final concentration OD600 1.0 and contains pGD-NPL (OD600
0.5), Agrobacterium hybrid injection this life Tobacco Leaves of pGD-VSRs (OD600 0.3), in fluorescence microscopy after hot-house culture 5-20 days
The RFP fluorescence (Fig. 6 A) that unicellular can be observed under mirror shows that the BYSMV of overall length has carried out duplication transcription in this life cigarette
Process.The club shaped structure similar to virion is further observed under an electron microscope, shows the virus in this life cigarette
(Fig. 6 B) can successfully be assembled.
D.BYSMV overall length infectious clone pCB-BYSMV-RFP is established in plant hopper to be infected: weighing injection areas this life tobacco leaf
Piece 2-3g is placed in alcohol calcination in advance and sterilizes and in the mortar that is pre-chilled on ice, 2ml Viral extraction Buffer grinding is added
(100mM Tris-HCl, 10mM Mg (CH3COO)2, 1mM MnCl2, 40mM Na2SO3, pH 6-9).After grinding, by viral juice
Liquid is transferred in 1.5ml pipe, 4 degree of centrifugation 10min of 12000g, and supernatant is viral crude extract.If crude extract is injected plant hopper
In polypide.Single sample injects 100 or so plant hopper nymphs, and injection is placed on rice seedling and is incubated for, observe every other day there are about
80% survives, and observes in 20% or so plant hopper body there is RFP fluorescence under body formula fluorescence microscope after 10-20 days, shows these
Plant hopper success is infected by BYSMV-RFP recombinant virus, as a result as shown in Figure 7.
Infectivity of the E.BYSMV overall length infectious clone pCB-BYSMV-RFP in barley: by flying with RFP fluorescence
After lice is incubated for 10-20 days, it is inoculated with healthy barley, barley can be observed after 10-20 days and appear similar to BYSMV wild virus
Symptom after infecting.It is able to observe that in morbidity Barley Cells there is RFP protein expression (Fig. 8), the above knot under fluorescence microscope
Fruit shows that the infectious cDNA clone that building obtains can successfully infect the host plants such as barley, generates biologically active
BYSMV virus.
4, BYSMV expression vector establishment and application
The building of A.pCB-BYSMV-EGFP expression vector skeleton:
Step 1: with primer primer19, primer25 linearization plasmid pBYMR segment, with primer primer34,
The EGFP segment of primer35 amplification, two segments are recombinated, and plasmid pBYMR-EGFP, in this carrier, EGFP two are obtained
End introduces MluI restriction enzyme site respectively；
Step 2: with primer primer30, primer31 by template amplification N 3U-EGFP-P 5U of pBYMR-EGFP it
Between sequence recombinate to the carrier pBYSNP-T linearized through primer primer32, primer33, obtain plasmid pBYSNP-
EGFP-T；
Step 3: expanding the BYSMV containing EGFP from plasmid pBYSNP-EGFP-T with primer primer28, primer29
Sequence is recombinated to the carrier pCB-BYSMV linearized through NotI, ClaI by seamless Cloning Kit and obtains recombinant vector pCB-
BYSMV-EGFP, constructs process and carrier schematic diagram is as shown in Figure 9.The recombinant virus carries EGFP reporter gene, can use length
The incidence (Figure 10) of wave ultraviolet lamp monitoring infection plant, it is often more important that EGFP can be cut with endonuclease MluI
Get off to replace with other purposes gene.
The application of B.pCB-BYSMV-EGFP expression vector skeleton:
In order to show expression vector expression clip size capacity, selected GUS (1812 bp) gene as external source table
The target fragment reached.
Firstly, expanding GUS sequence using primer primer36, primer37, it is recombined into the pCB- linearized through MluI
BYSMV-EGFP carrier obtains pCB-BYSMV-GUS recombinant plasmid.Plasmid pCB-BYSMV-GUS is converted into Agrobacterium competence
After EHA105 with include plasmid pGD-NPL and pGD-VSRs Agrobacterium co-injection this life Tobacco Leaves in, about 5-20 days
Taking injection areas blade to carry out GUS dyeing after 15 days can be observed part cell presentation navy blue (Figure 11), show that BYSMV takes
The gus gene of band successful expression in this life cigarette.Further using the method for D part and the part E in step 3, by pCB-
This life Tobacco Leaves of BYSMV-GUS morbidity are inoculated into healthy barley by plant hopper, and barley can be observed after 10-20 days and show disease, to whole
Strain morbidity barley carries out GUS dyeing and shows that root, stem, Ye Zhongjun have gus protein high abundance to accumulate (Figure 12).
The building of C.BYSMV dual-expression vector:
BYSMV dual-expression vector building, using primer primer19, primer25 linearization plasmid pBYMR, be recombined into through
The RFP segment of primer38, primer39 amplification obtains plasmid pBYMR-2, inserts BmtI digestion position at the both ends RFP in this way
Point.Using pBYMR-2 as template, with primer primer30, primer31 amplification N 3U-RFP (Bmt1)-P 5U sequence fragment recombination
The carrier pBYSNP-EGFP-T for entering primer primer32, primer40 linearisation obtains carrier pBYSNP-EGFP-RFP-T.Again
Then contain EGFP-RFP's using plasmid pBYSNP-EGFP-RFP-T as template amplification with primer primer28, vector-R
BYSMV segment is recombined into the pCB-BYSMV carrier linearized through NotI, ClaI double digestion, obtains plasmid pCB-BYSMV-
EGFP-RFP, wherein EGFP sequence both ends are MluI restriction enzyme sites, and the both ends RFP are BmtI restriction enzyme site, if replacement needs table
The target fragment reached can be added MluI or BmtI at the both ends of respective segments and replace EGFP or RFP (Figure 13).
The plasmid is transferred to after EHA105 Agrobacterium competence and includes the agriculture bar of plasmid pGD-NPL and pGD-VSRs
It can observe and same be simultaneously emitted by under fluorescence microscope into the cell in bacterium co-injection this life Tobacco Leaves, after about 5-20 days
GFP and RFP fluorescence, GFP the and RFP gene for showing that BYSMV virus carries can be expressed smoothly.By this life cigarette virus crude extract
It injects in plant hopper body, shows GFP, RFP fluorescence while being observed in plant hopper body under fluorescence microscope after 10-20 days
(Figure 14) can also observe simultaneously GFP and RFP fluorescence (Figure 15) after the virus is accessed barley by plant hopper in Barley Cells.
The building of D.BYSMV multiple representation carrier
The triple overexpressing protein vector constructions of BYSMV, the first step are with pCB-BYSMV using primer41, primer42
Template amplification P63U-M5U sequence fragment, which is connected, obtains plasmid pP63UM5U-T into pMD19T carrier, and PacI is added after M5U
Restriction enzyme site；Second step primer primer43, primer44 expand ECFP segment and are connected after restriction endonuclease Pac1 digestion with T4DNA
It connects enzyme and connect to obtain plasmid pBYSECFP-T with the P63UM5U-T carrier linearized through Pac1, remain with Pac1 at the both ends ECFP
Restriction enzyme site；Third step with primer primer45, primer46 using pCB-BYSMV the sequence fragment as between template amplification P-P3
It connects and obtains plasmid pPP3-T into pMD19T carrier；4th step using pBYSECFP-T as template using primer primer47,
Primer48 amplification P63U-M5U-ECFP segment is recombined into the carrier pPP3-T linearized through primer primer49, primer50
Plasmid pPP3ECFP-T is obtained afterwards；Finally using primer primer51, primer52 using plasmid pPP3ECFP-T as template amplification
P-ECFP-P3 sequence fragment is seamless be cloned into through ClaI, SwaI double digestion linearisation pCB-BYSMV-EGFP-RFP carrier obtain
To plasmid pCB-BYSMV-EGFP-RFP-ECFP, the both ends ECFP insert Pac1 restriction enzyme site to the carrier again again, can replace ECFP
It is changed to the target gene for wanting overexpression, while EGFP, RFP can also be replaced respectively and then express three albumen (Figure 16).
It is consistent with aforementioned process, by the plasmid be transferred to after EHA105 Agrobacterium competence with include plasmid pGD-NPL and
In Agrobacterium co-injection this life Tobacco Leaves of pGD-VSRs, it can be observed under fluorescence microscope after about 5-20 days same
A to be simultaneously emitted by GFP, RFP and CFP fluorescence into the cell, GFP, RFP and CFP gene for showing that BYSMV virus carries can be smooth
Expression.This life cigarette virus crude extract is injected in plant hopper body, can observe that plant hopper is intracorporal under fluorescence microscope after 10-20 days
GFP, RFP and CFP fluorescence are showed simultaneously, can also be observed simultaneously in Barley Cells after the virus is accessed barley by plant hopper
GFP, RFP and CFP fluorescence.
The application of E.BYSMV mediation gene editing carrier
The above result shows that BYSMV can accommodate biggish foreign gene insertion, therefore attempt to be transform as can be used for
The viral vectors of gene knockout.
It is inserted into cas9 albumen in the position of EGFP, guideRNA+gRNA scaffold is inserted into behind P.First with drawing
The pCB-BYSMV-EGFP carrier of object cas9-F, cas9-R amplification cas9 gene insertion Mlu1 linearisation obtains plasmid pCB-
BYSMV-CAS9；Then it is recombined into using primer primer53, primer54 amplification gRNA scaffold segment through vector-
The carrier pPP3-T of 11F, vector-11R linearisation obtains plasmid pPP3gs-T, in this way can be by connecting certainly after inverse PCR
It is merged before gRNA scaffold into guide RNA.This life cigarette 16C for stablizing and being transferred to GFP gene has been selected herein
(M.Teresa Ruiz et al., 1998) edits carrier as material to verify BYSMV.
First using pPP3gs-T as template, 20bp guide is inserted into primer primer55, primer54 inverse PCR
RNA is used to target mGFP, which, which is equipped with NdeI restriction enzyme site, can be used to digestion detection edited result；Then primer is used
The plasmid that primer56, primer57 are obtained using back is template amplification segment P- (GFP guide) gRNA scaffold-
P3 is recombined into the pCB-BYSMV-CAS9 carrier of NcoI linearisation；The plasmid is finally transferred to EHA105 Agrobacterium by the carrier
After competence with include plasmid pGD-NPL and pGD-VSRs Agrobacterium co-injection this life Tobacco Leaves in, about 10-15 days
The expression (Figure 17) of Cas9 albumen can be detected by western blot afterwards.
CTAB method extracts injection this life cigarette 16c injection blade and non-treated control group 16c leaf DNA, takes 2000ng, adds
37 DEG C of NdeI restriction endonuclease overnight digestions, are template primer primer58, primer59 through NEB high-fidelity using 2 μ L digestion products
Agarose electrophoresis detects PCR product after phusion archaeal dna polymerase expands 36 circulations.The result shows that injecting virus editor's carrier
DNA sample, can detect specific band at purpose size position, and comparison DNA sample is without band.Illustrate this position
The site NdeI cannot can normally be sheared by the target of gene editing and destruction by NdeI enzyme.And normal healthy controls, not by compiling
The NdeI restriction enzyme site for collecting the genome site of processing is normally cut into small fragment by NdeI enzyme.The following figure is sequencing result,
Further determine that different degrees of missing by gene editing target, and is caused in above-mentioned site.By DNA after purpose band gel extraction
Show that mGFP gene generates mutation (Figure 18) by guide RNA target mark region really after the sequencing of connection pMD-19T carrier afterwards.
It should be understood that after the dosage of above-described embodiment agents useful for same or raw material is carried out equal proportion expansion or is reduced
Technical solution, it is substantially identical with above-described embodiment.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>China Agricultural University
<120>plant cytoplasm rhabdovirus infectious clone, expression vector and its construction method and application
<141> 2018-05-09
<160> 75
<170> SIPOSequenceListing 1.0
<210> 1
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaagttcatt tcatttggag aggacgacca gtgatcgtat aatttgatta ttggtgatc 59
<210> 2
<211> 55
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ccggggatcc tctagagtcg actcatttat gagtatgata aaataccttt tctcg 55
<210> 3
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
gtattttatc atactcataa atgagtcgac tacaaagatt atagtaatta ataaaaact 59
<210> 4
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aggtggagat gccatgccga cccacgacca agtgagccgc aatctgtacc atc 53
<210> 5
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
cagatctcga 10
<210> 6
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gctcaaatgg 10
<210> 7
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
caaaagaaga 10
<210> 8
<211> 6
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tcatgg 6
<210> 9
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
gtcgactgca 10
<210> 10
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
gaattcgatt 10
<210> 11
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
aggagaagat 10
<210> 12
<211> 8
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ctggtcag 8
<210> 13
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cagatctcga 10
<210> 14
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
gctcaaatga 10
<210> 15
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
gctcatccaa 10
<210> 16
<211> 6
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
tgctgc 6
<210> 17
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gtcgactgca 10
<210> 18
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
gaattcgatt 10
<210> 19
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
agagatctcc 10
<210> 20
<211> 9
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ataaggatc 9
<210> 21
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ttcctcagat ctcgagctca atggatcttc tcgaagatga tgtc 44
<210> 22
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
ggtaccgtcg 10
<210> 23
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 23
actgcagaat 10
<210> 24
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 24
tcgatcaata 10
<210> 25
<211> 10
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 25
gggagttgta 10
<210> 26
<211> 8
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 26
aatgatgc 8
<210> 27
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 27
ccttatctgg gaactactca cacat 25
<210> 28
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
gcgcgctata ttttgttttc t 21
<210> 29
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
aaacaaaata tagcgcgcct cacacattat tatggagaa 39
<210> 30
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cgcgcgataa tttatcctag ttt 23
<210> 31
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
taggataaat tatcgcgcgc gaattcgagc tccaccgcg 39
<210> 32
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
gtttaattcc cgatctagta acatagatga cac 33
<210> 33
<211> 28
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
tactagatcg ggaattaaac tatcagtg 28
<210> 34
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
agttcccaga taagggaatt agggttc 27
<210> 35
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
ccatagtata aataataaaa acc 23
<210> 36
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 36
ttagagatct ccataaggat c 21
<210> 37
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 37
aggaatcggg aatcattaat atcta 25
<210> 38
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 38
ctttctggaa acgtcaaaaa tagaaacag 29
<210> 39
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 39
tatttttgac gtttccagaa agatgggtaa aggagaagaa cttttc 46
<210> 40
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 40
gatattaatg attcccgatt cctttagagt ccggacttgt atagttc 47
<210> 41
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 41
attgaccaag atttgaaatt cgaac 25
<210> 42
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 42
atttcaaatc ttggtcaata tggcctcctc cgagaacgtc atc 43
<210> 43
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 43
gtttttatta tttatactat ggttatctag atccggtgga tcccgg 46
<210> 44
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 44
gaggtatcca caacgccggc ggccgcggtg tctcgcacac ggcttcg 47
<210> 45
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 45
ccctgggccc agcttgcaat cgatgaaggt ctggaagcac tttctac 47
<210> 46
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 46
gaccagatct tctcctaaag gaatcgggaa tcattaata 39
<210> 47
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 47
taatgattcc cgattccttt atctagatcc ggtggatcc 39
<210> 48
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 48
aggaatcggg aatcattaat at 22
<210> 49
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 49
ttaggagaag atctggtcag c 21
<210> 50
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 50
cgaatttcaa atcttggtca atacgcgtat ggtgagcaag ggcgagg 47
<210> 51
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 51
atcataatgg tttttattat ttatactatg gacgcgttta cttgtacagc tcgtccat 58
<210> 52
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 52
tcgaatttca aatcttggtc aatacgcgta tggtccgtcc tgtagaaacc c 51
<210> 53
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 53
gatattaatg attcccgatt cctacgcgtt cattgtttgc ctccctgctg c 51
<210> 54
<211> 46
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 54
cgaatttcaa atcttggtca atgctagcat ggcctcctcc gagaac 46
<210> 55
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 55
tatcataatg gtttttatta tttatactat gggctagctt acaggaacag gtggtggcg 59
<210> 56
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 56
acgcgtttac ttgtacagct c 21
<210> 57
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 57
gaccccttaa ccgagtaact ccc 23
<210> 58
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 58
ttaattaatt tgtctgtcta tgttggtctc ag 32
<210> 59
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 59
ttaattaaat ggtgagcaag ggcgaggac 29
<210> 60
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 60
ttaattaatc atacctttct cttctttttt ggcttgtaca gctcgtccat g 51
<210> 61
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 61
atgagctcat ccaatgctgc a 21
<210> 62
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 62
gatgctgata gcccttgatt aa 22
<210> 63
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 63
catgatccct atggagatct ctaagacccc ttaaccgagt aac 43
<210> 64
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 64
atagctagat agattgatga tagatttaat taatcatacc tttctcttct tttttg 56
<210> 65
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 65
atctatcatc aatctatcta gctatcc 27
<210> 66
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 66
ttagagatct ccatagggat catg 24
<210> 67
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 67
gaaagtgctt ccagaccttc a 21
<210> 68
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 68
agttttgaag actgaatgag ggat 24
<210> 69
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 69
aataaataat aaaaactgag accaatgttt tagagctaga aatagcaagt 50
<210> 70
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 70
ccgcttgttg tccatttagg tctcctgttt ttagcaccga ctcggtgcca cttt 54
<210> 71
<211> 53
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 71
tcgtgccgct tcatatgatc gttttagagc tagaaatagc aagttaaaat aag 53
<210> 72
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 72
gcttactgca ggaagatgga atc 23
<210> 73
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 73
ctaaacaact aacaacaatt tctccatgg 29
<210> 74
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 74
atgagtaaag gagaagaact tttcac 26
<210> 75
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 75
gttgataatg atcagcgagt tgc 23
Claims (10)
1. a kind of plant cytoplasm Rhabdovirus expression vectors, which is characterized in that the expression vector is to contain barley Huang item point
The carrier of mosaic virus full-length genome reverse complementary strand.
2. the construction method of expression vector described in claim 1, which is characterized in that the genome of barley Huang item point mosaic virus
RNA carries out reverse transcription and PCR amplification, and the reverse complementary strand of resulting geneome RNA is cloned into containing cauliflower mosaic virus
In the plant expression vector of 35S promoter and Hepatitis C virus nucleic acid enzyme sequence.
3. construction method according to claim 2, which is characterized in that by the geneome RNA of barley Huang item point mosaic virus
After reverse transcription, it is divided into two sections or three sections and expands and be connected into plant expression vector.
4. construction method according to claim 2 or 3, which is characterized in that by the way of digestion connection or homologous recombination
The normal chain cDNA is building up in carrier.
5. a kind of infectious clone, which is characterized in that the infectious clone by (1) below mediated by agriculture bacillus~(2) or (1)~
(3) carrier described in infects gained after this life cigarette altogether:
(1) expression vector described in claim 1；
(2) carrier of N, P, L of BYSMV are expressed respectively；Or the carrier of N, P, L of BYSMV are expressed simultaneously；
(3) simultaneously expressing gene silencing suppressor p19, HCpro and γ b carrier.
6. a kind of method that worm passes plant virus infectious clone, which is characterized in that by infectivity gram described in claim 5
It is grand, through plant hopper insect as vector, infect monocotyledon.
7. the infectious clone in the monocotyledon obtained using claim 6 the method.
8. application of the expression vector described in claim 1 in terms of infecting monocotyledon or plant hopper insect, which is characterized in that
Monocotyledon or plant hopper insect and expression alien gene, the external source are infected using the expression vector foreign gene-carrying
Mrna length is within 5000bp, and the foreign gene is 1-3.
9. application according to claim 8, which is characterized in that the foreign gene is connected by homologous recombination or digestion
Mode is inserted between the N of expression vector and P or between P and P3.
10. application of the expression vector described in claim 1 in terms of carrying out gene editing to plant hopper or monocotyledon, feature
It is, Cas9 albumen and gRNA, or expression other types gene editing element can be expressed simultaneously using the expression vector.
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| PCT/CN2018/121425 WO2019223295A1 (en) | 2018-05-21 | 2018-12-17 | Plant cytorhabdovirus infectious clone, expression vector, and construction method therefor and application thereof |
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| WO2021129895A3 (en) * | 2019-12-23 | 2021-08-19 | 浙江大学 | Infectious plant rhabdovirus vector and method for non-transgenic, site-directed editing of plant genome |
| CN114317595A (en) * | 2021-12-29 | 2022-04-12 | 西南大学 | Rape mosaic virus infectious clone expression vector and construction method thereof |
| CN115216486A (en) * | 2021-04-21 | 2022-10-21 | 浙江大学 | Negative strand RNA viral vector and plant genome editing method without transformation |
| CN115397997A (en) * | 2020-12-23 | 2022-11-25 | 浙江大学 | Infectious plant rhabdovirus vector and method for site-specific editing of transgene-free plant genome |
| GB2607743A (en) * | 2019-12-23 | 2022-12-14 | Univ Zhejiang | Infectious plant rhabdovirus vector and method for non-transgenic, site-directed editing of plant genome |
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| CN111440818B (en) * | 2020-02-17 | 2022-06-10 | 南京农业大学 | Soybean mosaic virus infectious cloning vector capable of infecting tobacco, agrobacterium strain and application thereof |
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| CN104962580B (en) * | 2015-06-30 | 2018-04-27 | 浙江大学 | A kind of recombinant plant Rhabdovirus vector and its construction method |
| CN106521028B (en) * | 2016-11-07 | 2019-11-29 | 中国农业科学院植物保护研究所 | A kind of multiplex RT-PCR method for 4 kinds of arbovirus of synchronous detection |
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Non-Patent Citations (1)
| Title |
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| 严滕: "大麦黄条点花叶病毒基因组分析及磷酸化对磷蛋白功能的调控研究", 《中国博士学位论文全文数据库农业科技辑》 * |
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| WO2021129895A3 (en) * | 2019-12-23 | 2021-08-19 | 浙江大学 | Infectious plant rhabdovirus vector and method for non-transgenic, site-directed editing of plant genome |
| GB2607743A (en) * | 2019-12-23 | 2022-12-14 | Univ Zhejiang | Infectious plant rhabdovirus vector and method for non-transgenic, site-directed editing of plant genome |
| CN115397997A (en) * | 2020-12-23 | 2022-11-25 | 浙江大学 | Infectious plant rhabdovirus vector and method for site-specific editing of transgene-free plant genome |
| CN115216486A (en) * | 2021-04-21 | 2022-10-21 | 浙江大学 | Negative strand RNA viral vector and plant genome editing method without transformation |
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| CN115216486B (en) * | 2021-04-21 | 2024-04-16 | 浙江大学 | Negative strand RNA virus vector and plant genome editing method without transformation |
| CN114317595A (en) * | 2021-12-29 | 2022-04-12 | 西南大学 | Rape mosaic virus infectious clone expression vector and construction method thereof |
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