CN108588113A - Ternary shuttle vector and the method for building CYVCV infectious clones using it - Google Patents

Abstract

The invention discloses a kind of ternary shuttle vector pTY, sequence such as SEQ ID NO:Shown in 11.A kind of method using pTY structure Citrus Yellowing vein clearing virus (CYVCV) infectious clones is also disclosed, is included the following steps：(1) CYVCV full-length cDNAs are prepared；(2) restriction enzyme Stu I, Sma I digestions such as SEQ ID NO are used:The pTY of ternary shuttle vector shown in 11 obtains the carrier of pTY linearisations；(3) the adjoint homologous recombination of yeast conversion：Using Li-acetate method by linearized vector pTY and CYVCV full-length cDNA cotransformation saccharomycete YPH501；(4) infectious clone is identified：Homologous recombination plasmid converts Agrobacterium C58C1, and screening obtains CYVCV infectious clones.The present invention effectively overcomes virus full length cDNA segments and generates toxicity or unstable the phenomenon that leading to clone's failure in Escherichia coli.

Description

Ternary shuttle vector and the method for building CYVCV infectious clones using it

Technical field

The invention belongs to molecular biology field, it is related to a kind of ternary shuttle vector and builds CYVCV infectivities using its The method of clone.

Background technology

The Citrus Yellowing veinclearing disease caused by Citrus Yellowing vein clearing virus (CYVCV) is a kind of newfound citrus virus Disease.The disease is to be found on Pakistani lemon and bitter orange for 1988 earliest, and virus can cause lemon and bitter orange to occur The symptoms such as tender leaf veinclearing, yellow, blade warp and shrinkage can cause tender leaf to fall off, vein necrosis, tree vigo(u)r is caused to decline when serious Weak, fruit yield declines, even has no harvest sometimes.Grimaldi and Catara is in the lemonlike citrus leaf for yellow veinclearing symptom occur within 1996 On piece observes a kind of fibrous virus plastochondria, is then also observed in the different cultivars such as the citron of India, bitter orange and lemon It arrives, and is spread rapidly in Pakistan, Turkey.China found in 2009 on Ruili lemon for the first time, later in weight The ground such as celebrating, Sichuan, Jiangxi also find the disease in succession.CYVCV is α linear virals section (Alphaflexiviridae) India citrus The member of Tobamovirus (Mandarivirus), virion is linear, and a diameter of 13~14nm is about 685nm.CYVCV is just Adopted single strand RNA virus, the gene structure of CYVCV have certain stability, will not because of from different geographical difference host and Generate compared with Big mutation rate, it is now discovered that genome there are two types of type, one kind is 7529 nucleotide, and one kind is 7531 nucleotide, Including 5 ' and 3 ' end noncoding regions (UTR), 6 open reading frame (Openreading frame, ORFs) and Poly (A) tail. 6 open reading frame of CYVCV all originate in ATG, terminate at TAA (ORF1,4,5) or TGA (ORF2,3,6).

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, the report that there is no CYVCV infectious clones successfully to build both at home and abroad so far.Some researches show that utilize Escherichia coli structure When building larger geneome RNA virus full length cDNA clone, since the virus protein of its own coding may generate host strain Toxic effect recombinates so as to cause non-specificity, wild effect occurs.This becomes viral infectivity clone's structure and faces most Big difficult and challenge.The mechanism of virus full length genomic clone wild effect present in E.coli is still unclear.Usually It is solved using following method：By virus sequence cDNA clones, connection of short duration again before infecting；Or it is relatively low using copy number Carrier；Or regulate and control the environment (such as reducing cultivation temperature) of bacterial growth to reduce toxicity；Also have and utilize insertion introne Has virose albumen in viral whole genome sequence to avoid generating.But these methods take time and effort and success rate is low.

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 leaf spot virus as a case study.Virology Journal,2011,8(1):1-12) in structure apple chlorotic leaf spot virus (Apple chlorotic Leaf spot virus, ACLSV) when, from 36 through only identifying 1 in the correct E.coli clones of digestion with restriction enzyme A clone for having infectivity, shows to be cloned in E.coli cells by ACLSV that there is unstability, but obtains weight through yeast TAR technologies After group clone, the infectious clone of stable ACLSV is directly obtained by agriculture bacillus mediated inoculation.Arm spread moral wealth etc. (arm spread moral wealth, Shen Wentao, 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 invaded The new method tropical crops journals of metachromia clone, 2017,38 (8):1492-1500) in structure papaya deformity mosaic virus When (Papaya leaf distortion 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.In the trial of early period, CYVCV infectivities gram could not be obtained always by making host using Escherichia coli Grand, conjecture may be related with aforementioned unstability.

Invention content

It is an object of the invention in view of the above technical problems, provide a kind of ternary shuttle vector and utilize its structure The method of CYVCV infectious clones.

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

A kind of ternary shuttle vector pTY, nucleotide sequence such as SEQ ID NO:Shown in 11.

A method of structure CYVCV infectious clones include the following steps：

(1) CYVCV full-length cDNAs are prepared；

(2) restriction enzyme Stu I, Sma I digestions such as SEQ ID NO are used:Ternary shuttle vector shown in 11 PTY obtains the carrier of pTY linearisations；

(3) the adjoint homologous recombination of yeast conversion (TAR)：Using lithium acetate transformation method by the recycling of CYVCV full-length cDNAs The ternary shuttle vector pTY cotransformation saccharomycete YPH501 of segment and linearisation complete linearized vector pTY in yeast cells With the homologous recombination of CYVCV full-length cDNAs；

(4) infectious clone is identified：The yeast plasmid after homologous recombination is extracted, C58C1 or GV3101 is transferred to by electric shock In equal Agrobacteriums competent cell, Citrus Seedlings are inoculated with by agriculture bacillus mediated vacuum infiltration method, are examined in conjunction with conventional RT-PCR It surveys and Symptoms screening obtains CYVCV infectious clones.

The method that CYVCV full-length cDNAs are prepared in step (1) is long RT-PCR, and the primer is respectively such as SEQ ID NO:PTY-CYVCVF and pTY-CYVCVR shown in 3~4.

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

A, using restriction enzyme A or51H I (also known as Eco47III) single endonuclease digestion binary vector plasmid pTX1, line is obtained The carrier of property；

B, withPYES1LVector is template, with SEQ ID NO:PYES2117F shown in 1~2, PYES2117R is primer amplification, then recycles target fragment；

C, the recycling segment that pTX1 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 beneficial effects of the invention are as follows：Constructed ternary shuttle vector pTY cannot be only used for passing through in yeast cells Homologous recombination quick clone full length viral genome cDNA, and obtained recombinant plasmid can be posted through agriculture bacillus mediated direct inoculation Main plant goes out viral infectivity clone to Rapid identification, and pTY can also stablize duplication amplification in Escherichia coli, can be used for virus Full-length cDNA is sequenced.Using constructed ternary shuttle vector pTY, at home and abroad successfully constructs and be situated between suitable for Agrobacterium for the first time Connect the CYVCV infectious clones of kind.This method is by TAR technologies by virus full length cDNA and ternary shuttle expression carrier pTY After homologous recombination, Escherichia coli are not converted, but directly convert Agrobacterium C58C1, to obtain viral infectivity clone, effectively It overcomes virus full length cDNA segments and generates toxicity or unstable the phenomenon that leading to clone's failure in Escherichia coli.This method is logical It crosses agriculture bacillus mediated vacuum infiltration method and is directly inoculated with Citrus Seedlings, inoculation efficiency is high, and speed is fast.The method of the present invention can be in 1-2 It is realized in a month from long segment RT-PCR, TAR clone, Agrobacterium vacuum immersion to citrus and shows disease to obtain infectious clone Overall process, it is quick, efficient and of low cost.

Description of the drawings

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

Fig. 2 is CYVCV full-length genome one-step RT-PCR amplified production electrophoresis result figures, wherein M： DL15000DNAMarker；1~5：Sample.

Fig. 3 is the bacterium colony PCR qualification results of CYVCV full length cDNA clones, wherein M：2000bp molecular weight marker；1~ 8：CYVCV full-length cDNAs；9：Positive control；10：ddH₂O。

Fig. 4 is the symptom figure after agriculture bacillus mediated pTY-CYVCV inoculations citrus.

Fig. 5 is the symptom figure that agriculture bacillus mediated PTY-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.Double base table It is given up to vector plasmid pTX1 by professor Tao little Rong.Restriction enzyme Sac II, Stu I, Sma I, Aor51H I are purchased from north Capital NEB companies；LA Taqase、PrimerSTAR Max Premix、In-Fusion HD Cloning Kit、 PrimeScript^TMII 1st Strand cDNA Synthesis Kit, JM109 are purchased from TaKaRa companies；Trizol reagents,PYES1LVector is purchased from Invitrogen companies of the U.S..

The primer sequence is as shown in table 1 in the embodiment of the present invention：

1 primer sequence of table

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

Embodiment 1 builds ternary shuttle vector pTY

Using restriction enzyme A or51H I (also known as Eco47III) single endonuclease digestion binary vector plasmid pTX1, obtain linear The carrier of change.Endonuclease reaction system：Plasmid pTX112 μ L, restriction enzyme A or51H I (Eco47III) 2.5 μ L, 10 × 5 μ L of NEB Buffer, 23 μ L of distilled 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, reaction system：Linearisation pTX1 carriers add 2 μ L, insert Entering segment pYES1L-2117 adds 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 obtaining can be big in yeast-Agrobacterium- Stablize the ternary shuttle vector pTY replicated in enterobacteria (its plasmid map is shown in Fig. 1).PTY 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 elements left arm and right arm.Therefore, carrier pTY can be used for DNA fragmentation in yeast cells Homologous recombination and then recombinant DNA molecules are transformed into agrobacterium tumefaciens or Escherichia coli.In addition, working as virus full length cDNA When being cloned between StuI the and SmaI restriction enzyme sites of pTY, expression casette 2x35S-MCS-HDVRZ-NOS will ensure that Virus genomic accurate starting and termination.The nucleotide sequence of ternary shuttle vector pTY such as SEQ ID NO:11.

Embodiment 2CYVCV full-length cDNA amplifications

All steps of the present embodiment can refer to " amplification of the long RT-PCR of Citrus Yellowing vein clearing virus genome, gram Grand and sequence analysis " (Cui Tiantian etc., gardening journal, 2017,44 (5)：944–952.).

(1) Trizol (Invitrogen) method is used to extract citrus leaves total serum IgE.

(2) 5 ' RACE of CYVCV amplifications and clone identification：Detailed step is shown in the " long-chain of Citrus Yellowing vein clearing virus genome RT-PCR amplifications, Cloning and sequence analysis " (Cui Tiantian etc., gardening journal, 2017,44 (5)：944–952.).

(3) design of primers

According to having reported CYVCV whole genome sequences (GenBank accession number in NCBI：KP313240) and 5 ' are combined RACE expands the special primer of CYVCV gene orders using 5.0 Software for Design of PrimerPriemer.In design of primers, The overlapping fragments that 25~30bp is all generated between pTY carriers after pTY-CYVCVF and pTY-CYVCVR and digestion, in order to Homologous recombination is completed in yeast.Primer send to Invitrogen (Shanghai) Trading Co., Ltd. and synthesizes.

(4) CYVCV 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 CYVCV full-length cDNAs.

1) mixed liquor is prepared

2) program is run

Agarose gel electrophoresis result shows (Fig. 2) that PCR product clip size 7529bp meets expected size.

3. recycling purpose CYVCV full-length cDNA segments using DNA gel purification kit.

Embodiment 3 builds the infectious clone of CYVCV

(1) restriction enzyme Stu I, Sma I digested plasmid pTY are used, the carrier of pTY linearisations is obtained.Reactant System：1.0 μ L, 10 × NEB Buffer of plasmid pTY 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 of Stable 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 pTY of 10mg/mL Carrier 200ng, CYVCV full-length cDNA glue of linearisation recycles segment 200ng, and concussion keeps the component in transformation system fully mixed Even, 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 supernatant 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 to carry out SspI digestions to be accredited as the plasmid of overall length to identify CYVCV full length cDNA clones, be named as pTY- CYVCV。

In 20 clones of first picking, there are 10 overall length positive colonies, positive rate 50%.Second batch picking 12 clone in, have 9 overall length positive colonies, positive rate 75%.The average positive of CYVCV full length cDNA clones Rate is 65%.

(3) Agrobacterium-mediated Transformation and inoculation

CYVCV positive colony plasmids are extracted, by electroporated Agrobacterium C58C1, picking monoclonal connects bacterium to containing corresponding LB liquid medium (the 20mgL of antibiotic^-1Rif, 50mgL^-1Kan), 200rmin^-1, 28 DEG C of shaken cultivations 12~ 16h.Meanwhile breeding the Agrobacterium containing Hc-Pro or P19 expression plasmids.Thalline were collected by centrifugation, is suspended with inoculation buffer solution (outstanding Floating buffer solution：10mmol·L^-1MgCl₂, 10mmolL^-1MES, 200 μm of olL^-1As), make its OD₆₀₀Respectively 0.8~1.2 With 0.2~0.5, stands and be inoculated with this life cigarette using syringe after 2h.Using the Agrobacterium of pTY empty plasmids as negative control.

It is inoculated with brocade orange (C.sinensis) by agriculture bacillus mediated vacuum immersion, the symptom after being inoculated with 20 days and 40 days is seen Result is examined to show (Fig. 4)：There is the typical disease that the CYVCV such as veinclearing, yellow, distortion infect in the citrus leaves of infiltration pTY-CYVCV Shape, the symptom that blank control is then infected without performance CYVCV.The citrus leaves after being inoculated with 40 days are acquired, Plus methods is utilized to extract Blade total nucleic acid carries out conventional RT-PCR detection, as a result detects the specific band of 614bp, illustrates that pTY-CYVCV has and infects Property, CYVCV infectious clones are built successfully.

The present invention utilizes base on the basis of the successfully One step RT-PCR system of structure CYVCV Genomic full_length cDNAs In the viral infectivity cloned construct system of ternary shuttle vector pTY, at home and abroad obtains and connect suitable for agroinfiltration for the first time The CYVCV infectious clones of kind；The present invention does not convert large intestine by after viral genome and ternary shuttle expression carrier homologous recombination Bacillus, but Agrobacterium C58C1 is directly converted, effectively overcome the toxicity or unstable that large fragment generates in Escherichia coli Phenomenon；The CYVCV infectious clones that the present invention obtains are suitable for passing through agriculture bacillus mediated vacuum immersion citrus and injection inoculation The host plants such as cowpea, kidney bean.

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

The shuttle vector pTY in the present invention is infected for PVX (Potato VirusX) with reference to the method for preceding embodiment Property clone's structure, the amplifications of PVX full-length cDNAs uses primer pTY-PVXF, pTY-PVXR, yeast colony PCR detection to use primer PVX-F、PVX-R.It will be verified as positive pTY-PVX plasmids conversion Agrobacterium C58C1 and connect by Agrobacterium injection wetting method Kind this life cigarette, the observation result after being inoculated with 10 days are shown：Inject pTY-PVX tobacco symptom and positive control it is just the same, 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 methods is utilized to extract Blade total nucleic acid carries out conventional RT-PCR detection.The results show that the upper blade of pTY-PVX inoculations amplifies and expected size phase The purpose band (as shown in Figure 6) of symbol.This yeast recombinant clone system of explanation based on ternary shuttle vector pTY has good Applicability can be used for PVX and the infectious clone structure of other viruses.

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

The shuttle vector pTY 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 pTY has Applicability.

Sequence table

<110>Southwestern University

<120>Ternary shuttle vector and the method for building CYVCV infectious clones using it

<160> 11

<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> 59

<212> DNA

<213>Artificial sequence

<223> pTY-CYVCVF

<400> 3

atataaggaa gttcatttca tttggagagg agaaaagcaa acataaccaa cacacaccc 59

<210> 4

<211> 60

<212> DNA

<213>Artificial sequence

<223> pTY-CYVCVR

<400> 4

cgcgaggagg tggagatgcc atgccgaccc tttttttttt tttttttttt tttttcagaa 60

<210> 5

<211> 20

<212> DNA

<213>Artificial sequence

<223> CYVCV-F

<400> 5

taccgcagct atccatttcc 20

<210> 6

<211> 20

<212> DNA

<213>Artificial sequence

<223> CYVCV-R

<400> 6

gcagaaatcc cgaaccacta 20

<210> 7

<211> 56

<212> DNA

<213>Artificial sequence

<223> pTY-PVX-F

<400> 7

atataaggaa gttcatttca tttggagagg agaaaactaa accatacacc accaac 56

<210> 8

<211> 99

<212> DNA

<213>Artificial sequence

<223> pTY-PVX-R

<400> 8

cgcgaggagg tggagatgcc atgccgaccc gggttttttt tttttttttt tttttttttt 60

tttttttttt tttttttttt tttttttttt tttatttat 99

<210> 9

<211> 20

<212> DNA

<213>Artificial sequence

<223> PVX-F

<400> 9

atgtcagcac cagctagcac 20

<210> 10

<211> 23

<212> DNA

<213>Artificial sequence

<223> PVX-R

<400> 10

ggatccttat ggtggtggta gag 23

<210> 11

<211> 6783

<212> DNA

<213>Artificial sequence

<223>Ternary shuttle vector pTY

<400> 11

aagcttgcat gcctgcagtc aacatggtgg agcacgacac tctcgtctac tccaagaata 60

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 gacctgcagg 780

tcgactctag aggatccccg ggtcggcatg gcatctccac ctcctcgcgg tccgacctgg 840

gcatccgaag gaggacgtcg tccactcgga tggctaaggg agagctcgaa tttccccgat 900

cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 960

attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat gtaatgcatg 1020

acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 1080

atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 1140

ttactagatc ggaattcaga ttgtcgtttc ccgccttcag tttaaactat cagtgtttga 1200

caggatatat tggcgggtaa acctaagaga aaagagcgtt tattagaata atcggatatt 1260

taaaagggcg tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacagga 1320

gatctcagta aagcgctcct cgccgcagtt aattaaagtc agtgagcgag gaagcgcgta 1380

actataacgg tcctaaggta gcgaatcctg atgcggtatt ttctccttac gcatctgtgc 1440

ggtatttcac accgcataga tcggcaagtg cacaaacaat acttaaataa atactactca 1500

gtaataacct atttcttagc atttttgacg aaatttgcta ttttgttaga gtcttttaca 1560

ccatttgtct ccacacctcc gcttacatca acaccaataa cgccatttaa tctaagcgca 1620

tcaccaacat tttctggcgt cagtccacca gctaacataa aatgtaagct ttcggggctc 1680

tcttgccttc caacccagtc agaaatcgag ttccaatcca aaagttcacc tgtcccacct 1740

gcttctgaat caaacaaggg aataaacgaa tgaggtttct gtgaagctgc actgagtagt 1800

atgttgcagt cttttggaaa tacgagtctt ttaataactg gcaaaccgag gaactcttgg 1860

tattcttgcc acgactcatc tccatgcagt tggacgatat caatgccgta atcattgacc 1920

agagccaaaa catcctcctt aagttgatta cgaaacacgc caaccaagta tttcggagtg 1980

cctgaactat ttttatatgc ttttacaaga cttgaaattt tccttgcaat aaccgggtca 2040

attgttctct ttctattggg cacacatata atacccagca agtcagcatc ggaatctaga 2100

gcacattctg cggcctctgt gctctgcaag ccgcaaactt tcaccaatgg accagaacta 2160

cctgtgaaat taataacaga catactccaa gctgcctttg tgtgcttaat cacgtatact 2220

cacgtgctca atagtcacca atgccctccc tcttggccct ctccttttct tttttcgacc 2280

gaattaattc ttaatcggca aaaaaagaaa agctccggat caagattgta cgtaaggtga 2340

caagctattt ttcaataaag aatatcttcc actactgcca tctggcgtca taactgcaaa 2400

gtacacatat attacgatgc tgttctatta aatgcttcct atattatata tatagtaatg 2460

tcgtgatcta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 2520

ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 2580

ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 2640

accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 2700

gataataatg gtttcttaga cggatcgctt gcctgtaact tacacgcgcc tcgtatcttt 2760

taatgatgga ataatttggg aatttactct gtgtttattt atttttatgt tttgtatttg 2820

gattttagaa agtaaataaa gaaggtagaa gagttacgga atgaagaaaa aaaaataaac 2880

aaaggtttaa aaaatttcaa caaaaagcgt actttacata tatatttatt agacaagaaa 2940

agcagattaa atagatatac attcgattaa cgataagtaa aatgtaaaat cacaggattt 3000

tcgtgtgtgg tcttctacac agacaaggtg aaacaattcg gcattaatac ctgagagcag 3060

gaagagcaag ataaaaggta gtatttgttg gcgatccccc tagagtcttt tacatcttcg 3120

gaaaacaaaa actatttttt ctttaatttc tttttttact ttctattttt aatttatata 3180

tttatattaa aaaatttaaa ttataattat ttttatagca cgtgatgaaa aggacccagg 3240

tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 3300

aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaaa 3360

aaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 3420

ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 3480

gggacgcgta gtctagaggc tgaaccccca gccggaactg accccacaag gccctagcgt 3540

ttgcaatgca ccaggtcatc attgacccag gcgtgttcca ccaggccgct gcctcgcaac 3600

tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga atccgatccg 3660

cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga gctgaaatag 3720

tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat gaatttcgtg 3780

tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg gcaacgggac 3840

gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga ttccaggtgc 3900

ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag gcattcctcg 3960

gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag ctcgtagaac 4020

gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac ctgctgccac 4080

accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt cacgtccttg 4140

ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt gttgcgcgtg 4200

gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc cggccacggc 4260

gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg tttcagcaac 4320

gcggcctgct tggcctcgct gacctgtttt gccaggtcct cgccggcggt ttttcgcttc 4380

ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc tgccgcctcc 4440

tgttcgagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc agggggagcc 4500

agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggaccatcga gccgacggac 4560

tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc ggcatcctcg 4620

gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa cgtccgattc 4680

attcaccctc cttgcgggat tgccccgact cacgccgggg caatgtgccc ttattcctga 4740

tttgacccgc ctggtgcctt ggtgtccaga taatccacct tatcggcaat gaagtcggtc 4800

ccgtagaccg tctggccgtc cttctcgtac ttggtattcc gaatcttgcc ctgcacgaat 4860

accagcgacc ccttgcccaa atacttgccg tgggcctcgg cctgagagcc aaaacacttg 4920

atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat cgttgcgcca catctaggta 4980

ctaaaacaat tcatccagta aaatataata ttttattttc tcccaatcag gcttgatccc 5040

cagtaagtca aaaaatagct cgacatactg ttcttccccg atatcctccc tgatcgaccg 5100

gacgcagaag gcaatgtcat accacttgtc cgccctgccg cttctcccaa gatcaataaa 5160

gccacttact ttgccatctt tcacaaagat gttgctgtct cccaggtcgc cgtgggaaaa 5220

gacaagttcc tcttcgggct tttccgtctt taaaaaatca tacagctcgc gcggatcttt 5280

aaatggagtg tcttcttccc agttttcgca atccacatcg gccagatcgt tattcagtaa 5340

gtaatccaat tcggctaagc ggctgtctaa gctattcgta tagggacaat ccgatatgtc 5400

gatggagtga aagagcctga tgcactccgc atacagctcg ataatctttt cagggctttg 5460

ttcatcttca tactcttccg agcaaaggac gccatcggcc tcactcatga gcagattgct 5520

ccagccatca tgccgttcaa agtgcaggac ctttggaaca ggcagctttc cttccagcca 5580

tagcatcatg tccttttccc gttccacatc ataggtggtc cctttatacc ggctgtccgt 5640

catttttaaa tataggtttt cattttctcc caccagctta tataccttag caggagacat 5700

tccttccgta tcttttacgc agcggtattt ttcgatcagt tttttcaatt ccggtgatat 5760

tctcatttta gccatttatt atttccttcc tcttttctac agtatttaaa gataccccaa 5820

gaagctaatt ataacaagac gaactccaat tcactgttcc ttgcattcta aaaccttaaa 5880

taccagaaaa cagctttttc aaagttgttt tcaaagttgg cgtataacat agtatcgacg 5940

gagccgattt tgaaaccaca attatgggtg atgctgccaa ctcgagagcg ggccgggagg 6000

gttcgagaag ggggggcacc ccccttcggc gtgcgcggtc acgcgcacag ggcgcagccc 6060

tggttaaaaa caaggtttat aaatattggt ttaaaagcag gttaaaagac aggttagcgg 6120

tggccgaaaa acgggcggaa acccttgcaa atgctggatt ttctgcctgt ggacagcccc 6180

tcaaatgtca ataggtgcgc ccctcatctg tcagcactct gcccctcaag tgtcaaggat 6240

cgcgcccctc atctgtcagt agtcgcgccc ctcaagtgtc aataccgcag ggcacttatc 6300

cccaggcttg tccacatcat ctgtgggaaa ctcgcgtaaa atcaggcgtt ttcgccgatt 6360

tgcgaggctg gccagctcca cgtcgccggc cgaaatcgag cctgcccctc atctgtcaac 6420

gccgcgccgg gtgagtcggc ccctcaagtg tcaacgtccg cccctcatct gtcagtgagg 6480

gccaagtttt ccgcgaggta tccacaacgc cggcggccgc ggtgtctcgc acacggcttc 6540

gacggcgttt ctggcgcgtt tgcagggcca tagacggccg ccagcccagc ggcgagggca 6600

accagcccgg tgagcgtcta gtggactgat gggctgcctg tatcgagtgg tgattttgtg 6660

ccgagctgcc ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac 6720

aaattgacgc ttagacaact taataacaca ttgcggacgt ttttaatgta ctggggtggt 6780

ttt 6783

Claims (4)

1. a kind of ternary shuttle vector pTY, which is characterized in that its nucleotide sequence such as SEQ ID NO:Shown in 11.

2. a kind of method of structure Citrus Yellowing vein clearing virus infectious clone, which is characterized in that include the following steps：

(1) CYVCV full-length cDNAs are prepared；

(2) restriction enzyme Stu I, Sma I digestions such as SEQ ID NO are used:Ternary shuttle vector pTY, obtains shown in 11 The carrier linearized to pTY；

(3) the adjoint homologous recombination of yeast conversion：Using lithium acetate transformation method by the recycling segment of CYVCV full-length cDNAs and linearly It is complete to complete linearized vector pTY and CYVCV in yeast cells by the ternary shuttle vector pTY cotransformation saccharomycete YPH501 of change The homologous recombination of long cDNA；

(4) infectious clone is identified：The yeast plasmid after homologous recombination is extracted, the agricultures such as C58C1 or GV3101 are transferred to by electric shock In bacillus competent cell, screening obtains CYVCV infectious clones.

3. the method for structure CYVCV infectious clones as claimed in claim 2, which is characterized in that prepared in step (1) The method of CYVCV full-length cDNAs is long RT-PCR, and the primer is respectively such as SEQ ID NO:PTY- shown in 3~4 CYVCVF and pTY-CYVCVR.

4. the method for structure CYVCV infectious clones as claimed in claim 2, which is characterized in that three described in step (2) The construction method of first shuttle vector pTY is：

A, using restriction enzyme A or51H I single endonuclease digestion binary vector plasmid pXT1, the carrier linearized；

B, withPYES1LVector is template, with SEQ ID NO:PYES2117F, PYES2117R shown in 1~2 For primer amplification, target fragment is then recycled；

C, the recycling segment for obtaining linearized vector pXT1 and step B carries out recombination to construct, converts e. coli jm109, chooses Select positive colony to get.