CN108531502A

CN108531502A - The structure and inoculation method of citrus decline virus infectious clone

Info

Publication number: CN108531502A
Application number: CN201810367772.7A
Authority: CN
Inventors: 宋震; 崔甜甜; 宾羽; 晏建红; 李中安; 周常勇
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2018-04-23
Filing date: 2018-04-23
Publication date: 2018-09-14

Abstract

The invention discloses a kind of construction method of citrus decline virus infectious clone, steps：The long-chain RT PCR amplifications of (1) three covering CTV full-length genome specific fragments CTV1, CTV2, CTV3；(2) ternary shuttle vector pCY is linearized；(3) TAR is cloned：CTV1, CTV2, CTV3 are recycled to the ternary shuttle vector pCY cotransformation saccharomycete YPH501 of segment and linearisation using lithium acetate transformation method；(4) Agrobacterium is converted；(5) agriculture bacillus mediated inoculation obtains CTV infectious clones to identify.Also disclose the inoculation method of infectious clone：(1) it sows：Citrus Seeds peel off exosper and are sowed at culture medium, light culture；2) agrobatcerium cell for carrying CTV infectious clones and Hc Pro or P19 expression plasmids is collected by centrifugation, is suspended with inoculation buffer solution；(3) vacuum immersion；(4) light culture.

Description

The structure and inoculation method of citrus decline virus infectious clone

Technical field

The invention belongs to molecular biology field, it is related to structure and the inoculation side of a kind of citrus decline virus infectious clone Method.

Background technology

Citrus decline virus (Citrus tristezavirus, CTV) is that have to seriously endanger to the production of world's citrus A kind of pathogen.Brazil of world's citrus production second at present once once made its citrus industry be on the verge of to collapse because CTV is caused harm.So far Until the present, the citrus trees that CTV destroys still threaten in the world up to 100,000,000 plants or more using bitter orange as the citrus of stock and right The production of the kinds (being) such as grape fruit, shaddock and the certain sweet oranges of CTV stem trapping spot type strain sensitivities.In China, CTV distributions are more general Time, Preliminary Identification shows Strain distribution also very extensively.But due to China's m andarin and the upper common stock trifoliate orange of sweet orange production, acid How disease-resistant tangerine, red tangerine, red Canton lemon, Chinese holly head orange etc. be or resistance to sick and does not cause seriously to cause harm.Individual area such as Yunan border areas build water Make stock due to the use of susceptible citron and leads to Citrus Plants mortality.CTV can infect most kinds of both citrus, Cultigen and cenospecies can also infect both citrus some relative genus plants.CTV can be passed by the tissue graft comprising bast Broadcast, field by the aphids such as citrus aphid, cotten aphid, black citrus aphid and spiraea aphid by it is acyclic it is semi-durable in a manner of propagated.In addition, CTV can also be propagated by Semen Cuscutae.CTV can infect this life cigarette under artificial inoculation conditions.

CTV is A type Closteroviridae, and a kind of single-stranded monad RNA virus of justice of Closterovirus, is current Known maximum plant virus (Karasev et al., 1995).Its genome is the sense single stranded rna of 19226-19296nt, It is packaged in the closterovirus plastochondria of the helical symmetry of 2000 × 11nm, screw pitch 3.5-3.7nm, often encloses spiral by 8.5-10 Coat protein subunit is constituted.To T385 sequence analyses show CTV genomes include 12 opening code-reading frames (ORF1a, ORF1b, ) and 5 ' non-translation areas (5 ' UTR), 3 ' non-translation areas (3 ' UTR) ORF2-ORF11.

Infectious clone is not only that virus research provides the single and reliable genetic stocks of background, it may also be used for research virus Movement, duplication and its pathogenesis, while also be further investigation virus with host's Coupling effects, carry out viral vectors transformation Solid foundation is established with application.However, the constructing technology of virus full length cDNA infectious clones is stronger, construction work difficulty Greatly.Since CTV is the largest plant virus, so far, infectious clone only is obtained there are one T36 type CTV separation strains, it should Clone is built using traditional digestion connection method, and time-consuming and laborious and success rate is extremely low.Meanwhile the clone must be inoculated with this life first Cigarette, rear extraction virion can be inoculated with and infect citrus within 2 months or so, infect that the period is long, and infection rate is relatively low, also limit The application of the clone.In addition, some researches show that, when building larger geneome RNA virus full length cDNA clone using Escherichia coli, Since the virus protein of its own coding may generate toxic effect to host strain, recombinates, occur so as to cause non-specificity Wild effect.This becomes the greatest difficulty and challenge that viral infectivity clone's structure faces.Virus full length genomic clone exists The mechanism of wild effect present in E.coli is still unclear.Usually solved using following method：By virus sequence point Duan Kelong, connection of short duration again before infecting；Or utilize the copy lower carrier of number；Or the environment of regulation and control bacterial growth is (such as Reduce cultivation temperature etc.) reduce toxicity；Also have and have to avoid generating using in insertion introne to viral whole genome sequence Virose albumen.But these methods take time and effort and success rate is low.In the trial of early period, make host using Escherichia coli Most CTV separation strains all fail to obtain infectious clone, and conjecture may be related with aforementioned unstability.

The adjoint homologous recombination of yeast conversion (TAR) is a kind of to realize multiple phases using the efficient homologous recombination system of yeast Mutually there are the DNA fragmentation assemble methods of homologous sequence.Youssef etc. (YoussefF, Marais A, Faure C, Gentit P, Candresse T.Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs:Apple chlorotic leafspot virus as a case study.Virology Journal,2011,8(1):1-12) in structure apple chlorotic leaf spot virus (Apple chlorotic Leafspot virus, ACLSV) when, from 36 through only identifying 1 in the correct E.coli clones of digestion with restriction enzyme The clone for having infectivity, shows to be cloned in E.coli cells by ACLSV that there is unstability, but obtains recombination through yeast TAR technologies After clone, the infectious clone of stable ACLSV is directly obtained by agriculture bacillus mediated inoculation.(arm spread moral wealth, the Shen such as arm spread moral wealth Wen Tao, Yan Pu, Li little Ying, the yeast homologous recombination system of all E.coli-Free of roc a kind of are stablized rapid build PLDMV and are infected Property clone new method tropical crops journals, 2017,38 (8):1492-1500) in structure papaya deformity mosaic virus When (Papaya leafdistortion mosaic virus, PLDMV), it is found that there is also shakinesses in Escherichia coli by PLDMV Determine phenomenon, and when being inserted into introne in the P3 genes in PLDMV, it is correct to obtain sequencing by Transformed E .coli after yeast recombinates Stabilization infectious clone.

Invention content

It is an object of the invention in view of the above technical problems, provide a kind of structure of citrus decline virus infectious clone And inoculation method.

The present invention realizes that the technical solution of its purpose is：

A kind of construction method of citrus decline virus infectious clone, includes the following steps：

(1) the long RT-PCR amplification of CTV Genomic full_length cDNAs：The plant total serum IgE of extraction infection CTV, synthesizes its base Because of the first chain of group full-length cDNA, and then using three couples of special primers CTV1F, CTV1R, CTV2F, CTV2R and CTV3F, CTV3R divide PCR amplification is not carried out, obtains three specific fragments CTV1, CTV2, CTV3 of covering CTV full-length genomes；The primer CTV1F, CTV1R, CTV2F, CTV2R and CTV3F, CTV3R sequence respectively such as SEQ ID NO:Shown in 3~8；

(2) ternary shuttle vector pCY is linearized：Using restriction enzyme Stu I, Sma I digestions such as SEQ ID NO: Ternary shuttle vector pCY shown in 17 obtains the carrier of pCY linearisations；

(3) TAR is cloned：The ternary that CTV1, CTV2, CTV3 are recycled to segment and linearisation using lithium acetate transformation method shuttles Carrier pCY cotransformation saccharomycete YPH501 complete CTV full length cDNA clones in yeast cells by homologous recombination Structure；

(4) Agrobacterium is converted：The obtained yeast recombinant plasmid of extraction step (3) is transferred to C58C1 or GV3101 agricultures by electric shock Bacillus competent cell；

(5) agriculture bacillus mediated inoculation：It is inoculated with citrus or this life cigarette seedling, CTV infectious clones are obtained to identify.

The construction method of ternary shuttle vector pCY described in the step (2) is：

A, using restriction enzyme Sac II single endonuclease digestion binary expression vector plasmid DK1317-2, the load linearized Body；

B, withPYES1L Vector are template, such as SEQ ID NO:PYES2117F shown in 1~2, PYES2117R is primer amplification, then recycles target fragment；

C, the recycling segment that DK1317-2 carriers and step B are obtained will be linearized and carries out recombination to construct, convert Escherichia coli JM109, select positive colony to get.

The method of the agriculture bacillus mediated inoculation of the step (5) is inoculated with for vacuum infiltration method, or is connect by Agrobacterium injection CTV virion inoculation citrus is extracted after this life cigarette of kind 4-6 leaf phases again.

A kind of inoculation method of citrus decline virus infectious clone, using agriculture bacillus mediated vacuum infiltration method, specifically Include the following steps：

(1) it sows：Citrus Seeds peel off exosper, are sowed at light culture 5-10 days on culture medium；

(2) CTV is inoculated with buffer solution and prepares：It is collected by centrifugation respectively and carries CTV infectious clones and Hc-Pro or P19 expression matter The agrobatcerium cell of grain is suspended with inoculation buffer solution, makes its OD₆₀₀Respectively 0.8~1.2 and 0.2~0.5；

(3) vacuum immersion：The buffer solution that the obtained seedling of step (1) is immersed to step (2), is kept for 20~40 seconds under vacuum And it discharges rapidly；

(4) light culture：Inoculation is placed on incubator light culture 5~7 days, is transferred under natural conditions and cultivates.

The agrobatcerium cell of CTV infectious clone plasmids is carried in the step of above-mentioned inoculation method (2) to be wanted by right Step in 1 (1) to (4) is asked to obtain.

For including 10mmolL in the buffer solution for the agrobatcerium cell that suspends in the step of above-mentioned inoculation method (2)^- ¹MgCl₂, 10mmolL^-1MES, 200 μm of olL^-1AS。

The beneficial effects of the invention are as follows：The present invention builds CTV full-length genomes using the efficient homologous recombination system of yeast CDNA clone, effectively overcomes the limitation of restriction enzyme site in traditional digestion cascade synthesis, and entire regrouping process only needs a yeast to turn Change, can be obtained virus full length cDNA clone in 2 weeks, speed is fast, efficient；The invention will cover disease by TAR technologies After the segment and ternary shuttle expression carrier pCY homologous recombinations of virus gene group full-length cDNA, Escherichia coli are not converted, but directly Agrobacterium C58C1 is converted, to obtain viral infectivity clone, effectively overcomes full length viral genome cDNA in Escherichia coli Middle generation toxicity or unstable the phenomenon that leading to clone's failure, infectious clone pick-up rate significantly improves, up to 50% or so.It should Invention is directly inoculated with Citrus Seedlings by Agrobacterium vacuum infiltration method, avoids this life cigarette enriching virus necessary to traditional vaccination The process (the 1-2 months) of particle, inoculation efficiency significantly improves.In short, the present invention can realize in 1-2 months from CTV long segments RT- PCR, TAR clone, Agrobacterium vacuum immersion to citrus show disease to obtain the overall process of infectious clone, quickly, it is efficient and at This is cheap.

Description of the drawings

Fig. 1 is the plasmid map of ternary shuttle vector pCY.

Fig. 2 is a point result figure for three sections of CTV1, CTV2, CTV3 amplification CTV Genomic full_length cDNAs, wherein M： DL10000DNAMarker；1：Water；2~8：Sample.

Fig. 3 is the bacterium colony PCR qualification results of CTV full length cDNA clones, wherein M：2000bp molecular weight marker；1~5： CTV full-length cDNAs；6：ddH₂O；7：Positive control.

Fig. 4 is the symptom figure after agriculture bacillus mediated pCY-CTV inoculations citrus, wherein A, B：Infect type clone；C、D：It is unloaded Body compares.

Fig. 5 is the symptom figure that agriculture bacillus mediated PCY-PVX is inoculated with after this life cigarette.

Fig. 6 is the RT-PCR testing results of PVX, wherein M：DL2000DNAMarker；1~2：Sample；3：Positive control； 4：Negative control.

Specific implementation mode

Material therefor and reagent source of the present invention are as follows：

Yeast strain YPH501, agrobacterium strains C58C1 are given by French Thierry professors Candresse.

Restriction enzyme Sac II, Stu I, Sma I are purchased from Beijing NEB companies；LA Taqase、PrimerSTAR Max Premix、In-Fusion HD Cloning Kit、PrimeScript^TMII 1st Strand cDNA Synthesis Kit, JM109 are purchased from Dalian TaKaRa companies；Trizol reagents,PYES1LVector is purchased from the U.S. Invitrogen companies.

According to CTV whole genome sequences (GenBank accession number is EU937520) have been reported in NCBI, utilize 5.0 Software for Design of PrimerPriemer expands the special primer of CTV gene orders.CTV1F, CTV1R, CTV2F, CTV2R with CTV3F, CTV3R are respectively used for amplifying three specific fragments CTV1, CTV2, CTV3 of covering CTV full-length genomes； PYES2117F and PYES2117R is for expanding the segment for including yeast replication origin；PCY-PVX-F and pCY-PVX-R is used In amplification PVX overall lengths to verify the validity of shuttle vector.In design of primers, CTV1F and pCY carriers, the CTV1R after digestion With pCY carriers, the pCY- after pCY carriers, pCY-PVX-R and the digestion after CTV2F, CTV2R and CTV3F, CTV3R and digestion All there is the overlap of 25~32bp between pCY carriers after PVX-F and digestion respectively, it is homologous heavy in order to be completed in yeast Group.Primer is synthesized (table 1) by English fine horse (Shanghai) Bioisystech Co., Ltd.

1 design of primers of table and its sequence

Note：It is SacII restriction endonuclease recognition sequences in box, underscore part is the 30bp homologous with pCY carriers.

Embodiment 1 builds ternary shuttle vector pCY

Structure binary vector plasmid DK1317-2 first：It is transformed to obtain with plasmid PCMBIA1301 and pXT1, PCMBIA1301 is commercially obtained, and pXT1 carriers are given by Agricultural University Of Nanjing professor Tao little Rong.It is with pXT1 carriers Template includes pXT1 gene expression components (LB-2x35S-MCS-HDVRZ-NOS- with primer (TL1310F/TL1310R) amplification RB segment).PCAMBIA1301 plasmids are digested with PvuI.Then with gel extraction kit to including the amplification for being expected segment Recombination is purified and merged with digestion products.The VspI digestions of obtained fusion plasmid, big segment reconnect with structure Build pCAMBI-2x35S-MCS-HDVRZ-NOS, i.e. DK1317-2.

Using restriction enzyme Sac II single endonuclease digestion binary vector plasmid DK1317-2, the carrier linearized.Enzyme Cut reaction system：2.5 μ L, 10 × NEB Buffer of plasmid DK1317-212 μ L, restriction enzyme Sac II, 5 μ L, double steamings 23 μ L of water.Endonuclease reaction condition：37 DEG C of reaction 0.5h.

WithPYES1LVector is template, and PYES2117F, PYES2117R are that primer amplification contains yeast The segment pYES1L-2117 of related replication origin：Reaction volume is 25 μ L, including distilled water 8.5 μ L, PrimerSTAR Max Premix (2 ×) 12.5 μ L, specific each 1 μ L of upstream and downstream primer, templatepYES1LVector 1μL.Instead Answer condition：98 DEG C of 1min, 98 DEG C of 10s, 58 DEG C of 15s, 72 DEG C of 2min, 30 cycles；72 DEG C of 5min, 4 DEG C of preservations.Then it utilizes DNA gel purification kit recycles target fragment, is named as pYES1L-2117.

It is recombinated using In-Fusion HD Cloning Kit, system includes：Linearisation DK1317-2 carriers add 2 μ L, Insert Fragment pYES1L-2117 add 4 μ L, In-Fusion Enzyme to add 2 μ L, dd H₂O polishings are to 10 μ L.50 DEG C, water-bath 30min, 4 DEG C of preservations.E. coli jm109 is converted, 37 DEG C are incubated overnight, and select positive colony, and acquisition can be in yeast-agriculture bar The ternary shuttle vector pCY grown in bacterium-Escherichia coli (its plasmid map is shown in Fig. 1).PCY contains yeast (ARS4/CEN5), root The reproduction element of cancer agrobacterium (pVS1 or IV) and Escherichia coli (pBR322ori), expression casette 2x35S-MCS- HDVRZ-NOS is between T-DNA insertion elements left arm and right arm.Therefore, carrier pCY can be used for DNA pieces in yeast cells Section homologous assembling and then recombinant DNA molecules are transformed into Agrobacterium or Escherichia coli.In addition, working as virus full length cDNA When being cloned between StuI the and SmaI restriction enzyme sites of pCY, expression casette 2x35S-MCS-HDVRZ-NOS will ensure that Accurate starting and termination of the viral genome at exact site.The nucleotide sequence of ternary shuttle vector pCY is shown in SEQ ID NO:17。

The segmentation amplification of embodiment 2CTV Genomic full_length cDNAs

(1) Trizol methods are used to extract citrus leaves total serum IgE.

(2) design of primers

According to having reported CTV whole genome sequences (GenBank accession number in NCBI：EU937520) and combine 5 ' RACE, The special primer of CTV gene orders is expanded using 5.0 Software for Design of PrimerPriemer.In design of primers, CTV1F and enzyme PCY carriers, pCY-PVX-R and the enzyme after pCY carriers, CTV1R and CTV2F, CTV2R and CTV3F, CTV3R and digestion after cutting The overlap for all having 25~32bp between the pCY carriers after pCY carriers, pCY-PVX-F and digestion after cutting respectively, in order to Homologous recombination is completed in yeast.Primer send to Invitrogen (Shanghai) Trading Co., Ltd. and synthesizes.

(3) CTV full-length cDNA amplifications

1. the first chain synthesizes：Using the SuperScript of Invitrogen companies^TMII carries out the synthesis of the first chain cDNA.

Unwinding：

Run program：65 DEG C of denaturation 5min, set rapidly ice bath,

Room temperature prepares Buffer Mix

Run program：42 DEG C of incubation 2min

Finally it is separately added into 1 μ L (200U μ L^-1)SuperScript^TM, response procedures：42 DEG C of 50min, 70 DEG C of inactivations 15min, 4 DEG C of templates preserved as next step PCR reactions.

2. long-chain PCR reaction conditions：Using LATaq enzymes (TaKaRa companies), using 25 μ L reaction systems, 40 heat are followed Ring, to carry out the amplification of CTV full-length cDNAs.

1) mixed liquor is prepared:MI, M2, M3 be respectively used for amplifying covering CTV full-length genomes three specific fragment CTV1, CTV2、CTV3。

2) program is run

Agarose gel electrophoresis result shows (Fig. 2), PCR product clip size be respectively 7060bp, 6007bp, 6335bp meets expected size.

3. utilizing DNA gel purification kit recycling target fragment CTV1, CTV2, CTV3.

Embodiment 3 builds the infectious clone of CTV

(1) restriction enzyme Stu I, Sma I digested plasmid pCY are used, the carrier of pCY linearisations is obtained.Reactant System：1.0 μ L, 10 × NEB Buffer of plasmid pCY 13 μ L, restriction endonuclease sma I 5 μ L, 31 μ L of distilled water.Digestion is anti- Answer condition：Then 25 DEG C of reaction 0.5h add Stu I 1.0 μ L, 37 DEG C of incubation 0.5h.

(2) lithium acetate transformation method transformed yeast is utilized

With reference to (Tuo D, Shen W, Yan P, Li X, Zhou P.Rapid Construction ofStable such as Tuo Infectious Full-Length cDNA Clone of Papaya Leaf Distortion Mosaic Virus Using In-Fusion Cloning.Viruses,2015,7(12):6241-6250) and Youssef etc. (YoussefF, Marais A,Faure C,Gentit P,Candresse T.Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs:Apple chlorotic leafspot virus as a case study.Virology Journal,2011,8(1):1-12) use Lithium acetate transformation method, in the centrifuge tube for taking the competent yeast cells YPH501100 μ L to 2mL prepared, be sequentially added into Following reagent：36 μ L of LiAc of PEG4000 (50%w/v) 240 μ L, 1mol/L, salmon sperm dna 25 the μ L and pCY of 10mg/mL Carrier 200ng, CTV1, CTV2, CTV3 glue of linearisation recycles each 200ng of segment, and concussion keeps the component in transformation system abundant Mixing, 250r/min shakes bacterium 30min, 42 DEG C of water-bath heat shock 15min in 30 DEG C of shaking tables；6000r/min centrifuges 3min, discards Clear liquid uses 300 μ L ddH₂Cell is resuspended in O, is uniformly coated in Trp deficiency screening flat boards, and 2~4d is cultivated in 30 DEG C.

(3) yeast colony PCR detects

Wait for that yeast colony is grown to 1-3mm, with the 1/3 of toothpick picking colony, with 10 μ L ddH₂O suspends, 98 DEG C of processing 10min (broken wall) carries out PCR afterwards.The PCR reaction systems of use are as follows：

PCR response procedures are as follows：

Bacterium colony PCR qualification results show (Fig. 3) that obtained clip size is consistent with expected results.Positive colony further carries It takes plasmid and expands CTV1, CTV2, CTV3 respectively by preceding method to identify CTV full length cDNA clones, be named as pCY-CTV.In 20 clones of first picking, there are 7 positive full-length clones, positive rate 35%.Second batch picking 20 clones in, have 11 positive full-length clones, positive rate 55%.Two-wheeled independence TAR clone's gained CTV full-length genomes CDNA clone positive rate is 45%.(3) Agrobacterium-mediated Transformation and inoculation

CTV positive colony plasmids are extracted, Agrobacterium C58C1 is transferred to by electric shock.The inoculation of picking CTV positive monoclonals contains phase Answer the LB liquid medium (20mgL of antibiotic^-1Rif, 50mgL^-1Kan), 200rmin^-1, 28 DEG C of shaken cultivations 12~ 16h.Meanwhile the Agrobacterium monoclonal of the RNA silencing suppressors such as inoculated and cultured expression Hc-Pro or P19.Thalline were collected by centrifugation, With buffer solution suspension (buffer suspension liquid：10mmol·L^-1MgCl₂, 10mmolL^-1MES, 200 μm of olL^-1AS), make it OD₆₀₀It is spare after respectively 0.8~1.2 and 0.2~0.5, standing 2h.Meanwhile using the Agrobacterium of pCY empty plasmids as feminine gender Control.

Sowing is to MS culture mediums after brocade orange (C.sinensis) seed removes exosper, and light culture is after 5-10 days for being inoculated with. Seedling is immersed into above-mentioned inoculation buffer solution, vacuumize and is kept for 30 seconds, discharges rapidly, is placed in incubator light culture 7 days, is transferred to certainly It is cultivated under the conditions of so.The citrus leaves after being inoculated with 30 days are acquired, Plus methods extracting blade total nucleic acid is utilized to carry out conventional RT-PCR Detection.The results show that the upper blade of pCY-CTV inoculations amplifies the purpose band being consistent with expected 672bp sizes, infection rate 90%, illustrate that pCY-CTV is the clone for having infectivity.Symptom Observation result after being inoculated with 90 days is shown (Fig. 4)：Infiltrate pCY- There are the CTV such as veinclearing, yellow and infect symptom, the symptom that blank control is then infected without performance CTV in the citrus leaves of CTV.Into one Step illustrates that CTV infectious clones are built successfully.

The present invention builds CTV full length cDNA clones using the efficient homologous recombination system of yeast, effectively breaches The limitation of restriction enzyme site in traditional digestion cascade synthesis, entire regrouping process only need a yeast conversion, and it is complete to obtain CTV in 2 weeks Long cDNA clone；The invention is by TAR technologies by the segment of Corticovirus Genomic full_length cDNA and ternary shuttle expression carrier After pCY homologous recombinations, Escherichia coli are not converted, but directly convert Agrobacterium C58C1, to obtain viral infectivity clone, It effectively overcomes full length viral genome cDNA and generates toxicity or unstable the phenomenon that leading to clone's failure in Escherichia coli, invade Metachromia clone's pick-up rate significantly improves, and reaches 45%.The invention is directly inoculated with Citrus Seedlings by Agrobacterium vacuum infiltration method, keeps away The process (the 1-2 months) of this life cigarette enriching virus particles necessary to traditional vaccination is exempted from, inoculation efficiency significantly improves.In short, this Embodiment realizes from CTV long segments RT-PCR, TAR clone, Agrobacterium vacuum immersion to citrus in 2 months and shows disease to obtain The overall process of infectious clone, it is quick, efficient and of low cost.

4 shuttle vector pCY of embodiment is built for PVX infectious clones

With reference to preceding embodiment method by the present invention shuttle vector pCY and method be used for PVX (Potato Virus X) infectious clone is built, and the amplification of PVX full-length cDNAs uses primer pCY-PVXF, pCY-PVXR, yeast colony PCR detection to adopt With primer PVX-F, PVX-R.It will be verified as positive pCY-PVX plasmids conversion Agrobacterium C58C1 and inject by Agrobacterium to soak Profit method is inoculated with this life cigarette, and the observation result after being inoculated with 10 days is shown：Inject pCY-PVX tobacco symptom and positive control it is complete Equally, apparent comparison (as shown in Figure 5) is formed with negative control.This life cigarette tender leaf after being inoculated with 10 days is acquired, Plus is utilized Method extracts blade total nucleic acid and carries out conventional RT-PCR detection.The results show that the upper blade of pCY-PVX inoculations is amplified and is expected The purpose band (as shown in Figure 6) that size is consistent.This yeast recombinant clone system of explanation based on ternary shuttle vector pCY has Good applicability can be used for PVX and the infectious clone structure of other viruses.

5 shuttle vector pCY of embodiment is built for CLBV infectious clones

The shuttle vector pCY of the present invention is used for citrus leaf mottle virus (Citrus with reference to the method for preceding embodiment Leafblotch virus, CLBV) infectious clone structure.Pass through the efficiency for the CLBV full length cDNA clones that yeast recombination obtains 70% or more can be reached.It is good that this further illustrates that the yeast recombinant clone system based on ternary shuttle vector pCY has Applicability.

Sequence table

<110>Southwestern University

<120>The structure and inoculation method of citrus decline virus infectious clone

<160> 17

<210> 1

<211> 39

<212> DNA

<213>Artificial sequence

<223> PYES2117F

<400> 1

gccgattttg aaaccgcgga gtcagtgagc gaggaagcg 39

<210> 2

<211> 39

<212> DNA

<213>Artificial sequence

<223> PYES2117R

<400> 2

ctgcctgtga tcaccgcggc atcttttact ttcaccagc 39

<210> 3

<211> 52

<212> DNA

<213>Artificial sequence

<223> CTV1F

<400> 3

ctatataagg aagttcattt catttggaga ggaatttctc aaattcaccc gt 52

<210> 4

<211> 45

<212> DNA

<213>Artificial sequence

<223> CTV1R

<400> 4

taccacgaag accgggtgtg tcactaagca acgactcatc caact 45

<210> 5

<211> 47

<212> DNA

<213>Artificial sequence

<223> CTV2F

<400> 5

agttggatga gtcgttgctt agtgacacac ccggtcttcg tggtaac 47

<210> 6

<211> 44

<212> DNA

<213>Artificial sequence

<223> CTV2R

<400> 6

accgactacc actagcttca aactttgcga ctcgggcata ctag 44

<210> 7

<211> 44

<212> DNA

<213>Artificial sequence

<223> CTV3F

<400> 7

ctagtatgcc cgagtcgcaa agtttgaagc tagtggtagt cggt 44

<210> 8

<211> 74

<212> DNA

<213>Artificial sequence

<223> CTV3R

<400> 8

cgcgaggagg tggagatgcc atgccgaccc gggttttttt tttttttttt tttttttttt 60

tttggaccta tgtt 74

<210> 9

<211> 18

<212> DNA

<213>Artificial sequence

<223> CTV-CP3

<400> 9

tcaacgtgtg ttgaattt 18

<210> 10

<211> 18

<212> DNA

<213>Artificial sequence

<223> CTV-CP1

<400> 10

atggacgacg aaacaaag 18

<210> 11

<211> 20

<212> DNA

<213>Artificial sequence

<223> PVX-F

<400> 11

atgtcagcac cagctagcac 20

<210> 12

<211> 23

<212> DNA

<213>Artificial sequence

<223> PVX-R

<400> 12

ggatccttat ggtggtggta gag 23

<210> 13

<211> 41

<212> DNA

<213>Artificial sequence

<223> TL1310F

<400> 13

aactcgagct tgtcgattaa tgcatgcctg cagtcaacat g 41

<210> 14

<211> 33

<212> DNA

<213>Artificial sequence

<223> TL1310R

<400> 14

aaatgtttga acgatcgggg aaattcgagc tct 33

<210> 15

<211> 56

<212> DNA

<213>Artificial sequence

<223> pCY-PVX-F

<400> 15

atataaggaa gttcatttca tttggagagg agaaaactaa accatacacc accaac 56

<210> 16

<211> 99

<212> DNA

<213>Artificial sequence

<223> pCY-PVX-R

<400> 16

cgcgaggagg tggagatgcc atgccgaccc gggttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttatttat 99

<210> 17

<211> 10347

<212> DNA

<213>Artificial sequence

<223>Ternary shuttle vector pCY

<400> 17

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tcaaagatac agtctcagaa gaccagaggg ctattgagac ttttcaacaa agggtaatat 120

cgggaaacct cctcggattc cattgcccag ctatctgtca cttcatcgaa aggacagtag 180

aaaaggaaga tggcttctac aaatgccatc attgcgataa aggaaaggct atcgttcaaa 240

gaatgcctct accgacagtg gtcccaaaga tggacccccc acccacgagg aacatcgtgg 300

aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat aacatggtgg 360

agcacgacac tctcgtctac tccaagaata tcaaagatac agtctcagaa gaccagaggg 420

ctattgagac tttcaacaaa gggtaatatc gggaaacctc ctcggattcc attgcccagc 480

tatctgtcac ttcatcgaaa ggacagtaga aaaggaagat ggcttctaca aatgccatca 540

ttgcgataaa ggaaaggcta tcgttcaaga atgcctctac cgacagtggt cccaaagatg 600

gacccccacc cacgaggaac atcgtggaaa aagaagacgt tccaaccacg tcttcaaagc 660

aagtggattg atgtgatatc tccactgacg taagggatga cgcacaatcc cactatcctt 720

cgcaagaccc ttcctctata taaggaagtt catttcattt ggagaggcct atgagtaaag 780

gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg 840

ggtacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga aaacttaccc 900

ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt 960

tctcttatgg tgttcaatgc ttttcaagat acccagatca tatgaagcgg cacgacttct 1020

tcaagagcgc catgcctgag ggatacgtgc aggagaggac catcttcttc aaggacgacg 1080

ggaactacaa gacacgtgct gaagtcaagt ttgagggaga caccctcgtc aacaggatcg 1140

agcttaaggg aatcgatttc aaggaggacg gaaacatcct cggccacaag ttggaataca 1200

actacaactt ccacaacgta tacatcatgg ccgacaagca aaagaacggc atcaaagcca 1260

acttcaagac ccgccacaac atcgaagacg gcggcgtgca actcgctgat cattatcaac 1320

aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtccacac 1380

aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt cttgagtttg 1440

taacagctgc tgggattaca catggcatgg atgaactata caaataaccc gggtcggcat 1500

ggcatctcca cctcctcgcg gtccgacctg ggcatccgaa ggaggacgtc gtccactcgg 1560

atggctaagg gagagctcga atttccccga tcgttcaaac atttggcaat aaagtttctt 1620

aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt tgaattacgt 1680

taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg tttttatgat 1740

tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc gcgcaaacta 1800

ggataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa actatcagtg 1860

tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga 1920

tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac 1980

agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg 2040

gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta 2100

cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca 2160

taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg 2220

ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg 2280

ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc 2340

gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag 2400

tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca 2460

tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc 2520

cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa 2580

tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc 2640

cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag 2700

gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg 2760

cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg 2820

aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac 2880

aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga 2940

ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg 3000

gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt 3060

ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag 3120

cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg 3180

gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc 3240

gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc 3300

gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt 3360

gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc 3420

gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc 3480

gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg 3540

gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc 3600

gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg 3660

tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc 3720

gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag 3780

gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga 3840

gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa 3900

cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg 3960

aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc 4020

tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga 4080

attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg 4140

gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc 4200

cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc 4260

atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 4320

cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 4380

agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 4440

gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 4500

cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 4560

tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 4620

cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 4680

ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 4740

gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 4800

gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 4860

tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 4920

ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc 4980

tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 5040

cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 5100

cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 5160

cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 5220

ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 5280

caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 5340

aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 5400

tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 5460

cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 5520

cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 5580

gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 5640

cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 5700

cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 5760

gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac 5820

acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag 5880

cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac 5940

gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag 6000

agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 6060

gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 6120

ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 6180

aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 6240

ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 6300

gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 6360

cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 6420

gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 6480

tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 6540

cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 6600

cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 6660

gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 6720

agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 6780

cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 6840

tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 6900

tttggtcatg cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt 6960

ctcccaatca ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc 7020

gatatcctcc ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc 7080

gcttctccca agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc 7140

tcccaggtcg ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc 7200

atacagctcg cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc 7260

ggccagatcg ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt 7320

atagggacaa tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc 7380

gataatcttt tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc 7440

ctcactcatg agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac 7500

aggcagcttt ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt 7560

ccctttatac cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt 7620

atatacctta gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag 7680

ttttttcaat tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta 7740

cagtatttaa agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc 7800

cttgcattct aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg 7860

gcgtataaca tagtatcgac ggagccgatt ttgaaaccgc ggagtcagtg agcgaggaag 7920

cgcgtaacta taacggtcct aaggtagcga atcctgatgc ggtattttct ccttacgcat 7980

ctgtgcggta tttcacaccg catagatcgg caagtgcaca aacaatactt aaataaatac 8040

tactcagtaa taacctattt cttagcattt ttgacgaaat ttgctatttt gttagagtct 8100

tttacaccat ttgtctccac acctccgctt acatcaacac caataacgcc atttaatcta 8160

agcgcatcac caacattttc tggcgtcagt ccaccagcta acataaaatg taagctttcg 8220

gggctctctt gccttccaac ccagtcagaa atcgagttcc aatccaaaag ttcacctgtc 8280

ccacctgctt ctgaatcaaa caagggaata aacgaatgag gtttctgtga agctgcactg 8340

agtagtatgt tgcagtcttt tggaaatacg agtcttttaa taactggcaa accgaggaac 8400

tcttggtatt cttgccacga ctcatctcca tgcagttgga cgatatcaat gccgtaatca 8460

ttgaccagag ccaaaacatc ctccttaagt tgattacgaa acacgccaac caagtatttc 8520

ggagtgcctg aactattttt atatgctttt acaagacttg aaattttcct tgcaataacc 8580

gggtcaattg ttctctttct attgggcaca catataatac ccagcaagtc agcatcggaa 8640

tctagagcac attctgcggc ctctgtgctc tgcaagccgc aaactttcac caatggacca 8700

gaactacctg tgaaattaat aacagacata ctccaagctg cctttgtgtg cttaatcacg 8760

tatactcacg tgctcaatag tcaccaatgc cctccctctt ggccctctcc ttttcttttt 8820

tcgaccgaat taattcttaa tcggcaaaaa aagaaaagct ccggatcaag attgtacgta 8880

aggtgacaag ctatttttca ataaagaata tcttccacta ctgccatctg gcgtcataac 8940

tgcaaagtac acatatatta cgatgctgtt ctattaaatg cttcctatat tatatatata 9000

gtaatgtcgt gatctatggt gcactctcag tacaatctgc tctgatgccg catagttaag 9060

ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 9120

atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 9180

gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 9240

tgtcatgata ataatggttt cttagacgga tcgcttgcct gtaacttaca cgcgcctcgt 9300

atcttttaat gatggaataa tttgggaatt tactctgtgt ttatttattt ttatgttttg 9360

tatttggatt ttagaaagta aataaagaag gtagaagagt tacggaatga agaaaaaaaa 9420

ataaacaaag gtttaaaaaa tttcaacaaa aagcgtactt tacatatata tttattagac 9480

aagaaaagca gattaaatag atatacattc gattaacgat aagtaaaatg taaaatcaca 9540

ggattttcgt gtgtggtctt ctacacagac aaggtgaaac aattcggcat taatacctga 9600

gagcaggaag agcaagataa aaggtagtat ttgttggcga tccccctaga gtcttttaca 9660

tcttcggaaa acaaaaacta ttttttcttt aatttctttt tttactttct atttttaatt 9720

tatatattta tattaaaaaa tttaaattat aattattttt atagcacgtg atgaaaagga 9780

cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 9840

acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 9900

aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 9960

attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgccgcggt 10020

gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc cgcgagatca 10080

tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg gtaacatgag 10140

caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg atgggctgcc 10200

tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc tggctggtgg 10260

caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca cattgcggac 10320

gtttttaatg tactgaatta acgccga 10347

Claims

1. a kind of construction method of citrus decline virus infectious clone, which is characterized in that include the following steps：

(1) the long RT-PCR amplification of CTV Genomic full_length cDNAs：The plant total serum IgE of extraction infection CTV, synthesizes its genome The first chain of full-length cDNA, and then using three couples of special primer CTV1F, CTV1R, CTV2F, CTV2R and CTV3F, CTV3R respectively into Row PCR amplification obtains three specific fragments CTV1, CTV2, CTV3 of covering CTV full-length genomes；The primer CTV1F, CTV1R, CTV2F, CTV2R and CTV3F, CTV3R sequence respectively such as SEQ ID NO:Shown in 3~8；

(2) ternary shuttle vector pCY is linearized：Using restriction enzyme Stu I, Sma I digestions such as SEQ ID NO:17 institutes The ternary shuttle vector pCY shown obtains the carrier of pCY linearisations；

(3) TAR is cloned：CTV1, CTV2, CTV3 are recycled to the ternary shuttle vector of segment and linearisation using lithium acetate transformation method PCY cotransformation saccharomycete YPH501 complete the structure of CTV full length cDNA clones in yeast cells by homologous recombination It builds；

(4) Agrobacterium is converted：The obtained yeast recombinant plasmid of extraction step (3) is transferred to C58C1 or GV3101 Agrobacteriums by electric shock Competent cell；

2. the construction method of citrus decline virus infectious clone as described in claim 1, which is characterized in that in step (2) The construction method of the ternary shuttle vector pCY is：

A, using restriction enzyme Sac II single endonuclease digestion binary expression vector plasmid DK1317-2, the carrier linearized；

3. the construction method of citrus decline virus infectious clone as described in claim 1, which is characterized in that the step (5) method of agriculture bacillus mediated inoculation is inoculated with for vacuum infiltration method, or passes through this life cigarette of Agrobacterium injection inoculation 4-6 leaf phases Extract CTV virion inoculation citrus again afterwards.

4. a kind of inoculation method of citrus decline virus infectious clone, which is characterized in that use agriculture bacillus mediated Vaccum Permeating Profit method, specifically comprises the following steps：

(2) CTV is inoculated with buffer solution and prepares：It is collected by centrifugation respectively and carries CTV infectious clones and Hc-Pro or P19 expression plasmids Agrobatcerium cell is suspended with inoculation buffer solution, makes its OD₆₀₀Respectively 0.8~1.2 and 0.2~0.5；

(3) vacuum immersion：The obtained seedling of step (1) is immersed to the buffer solution of step (2), is kept for 20~40 seconds under vacuum and fast Quick-release is put；

5. the inoculation method of citrus decline virus infectious clone as claimed in claim 4, which is characterized in that the step (2) agrobatcerium cell that CTV infectious clone plasmids are carried in is obtained by step in claim 1 (1) to (4).

6. the inoculation method of citrus decline virus infectious clone as claimed in claim 4, which is characterized in that the step (2) for including 10mmolL in the buffer solution for the agrobatcerium cell that suspends in^-1MgCl₂, 10mmolL^-1MES, 200 μm of ol L^-1AS。