CN116064638A - E.coli-yeast-agrobacterium ternary shuttle vector and application thereof in plant virus infectious cloning - Google Patents

E.coli-yeast-agrobacterium ternary shuttle vector and application thereof in plant virus infectious cloning Download PDF

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CN116064638A
CN116064638A CN202211258827.3A CN202211258827A CN116064638A CN 116064638 A CN116064638 A CN 116064638A CN 202211258827 A CN202211258827 A CN 202211258827A CN 116064638 A CN116064638 A CN 116064638A
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yeast
agrobacterium
escherichia coli
shuttle vector
cmv
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张坤
郭枭
陈佳欢
贺振
顾天潇
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Yangzhou University
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Abstract

The invention belongs to the technical field of genetic engineering, and discloses an escherichia coli-yeast-agrobacterium shuttle vector and application thereof in plant virus infectious cloning. The escherichia coli-yeast-agrobacterium shuttle vector is based on the transformation of an escherichia coli-agrobacterium binary shuttle vector, namely, yeast DNA, replication initiation recognition sequences and yeast tryptophan synthase screening marker gene fragments are added, target gene fragments carrying a yeast replication element 2 mu sequence and a screening marker gene sequence are inserted into an escherichia coli-agrobacterium binary plasmid skeleton, two escherichia coli-yeast-agrobacterium ternary shuttle plasmids are constructed, and the plasmids can be stably inherited and proliferated in yeast cells, escherichia coli cells and agrobacterium cells.

Description

E.coli-yeast-agrobacterium ternary shuttle vector and application thereof in plant virus infectious cloning
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an escherichia coli-yeast-agrobacterium shuttle vector and application thereof.
Background
Agrobacterium mediation requires the use of binary vectors containing a T-DNA sequence that can be inserted into the plant genome by infecting plant or fungal cells. The binary vectors currently used are pCAMBIA series (such as pCAMBIA 1300), pBHt1, pSN1301, pUN1301, PSN1301 and the like. These binary vectors typically contain a DNA replicon of E.coli and a bacterial selection marker (e.g., a resistance gene such as Amp, kana, TET) together with a DNA replicon of Agrobacterium (e.g., pVS1_origin, pVS1-REP, etc.) and a T-DNA sequence. Thus, these binary vectors are E.coli-Agrobacterium shuttle vectors, which replicate and proliferate in E.coli and Agrobacterium. However, the above vector construction method is not suitable for high-throughput DNA transformation and gene knockout operations.
Yeast homologous recombination is a highly efficient, rapid and suitable method for constructing vectors with yeast replicon and yeast selection markers, which can be used for homologous recombination of DNA, and these vectors include pGBKT7, pYES2, pYIP5, pPICZ, pRS426 and the like, each containing a yeast DNA replicon (e.g., pGBK-R7 or 2micro 2_origin), a yeast selection marker gene (e.g., TRP1, URA3, LEU2, HIS3 and the like), and generally also containing E.coli DNA replicon and bacterial selection marker (e.g., amp, kana, TET and the like resistance genes). Thus, these yeast plasmids are E.coli-yeast shuttle vectors that replicate and proliferate in yeast and E.coli cells.
However, all of these E.coli-yeast shuttle vectors are currently unable to replicate and proliferate within the Agrobacterium cells and therefore cannot be used directly in Agrobacterium-mediated DNA transformation processes. The DNA fusion fragments (gene expression sequences, gene knockout structures, etc.) constructed by the yeast homologous recombination method need to be inserted into an E.coli-Agrobacterium shuttle vector after the target DNA fragments are excised from the yeast plasmid using restriction enzymes, and then used for Agrobacterium-mediated DNA transformation. However, the above-mentioned vector construction method has the disadvantages of very low efficiency and long time consumption, so that a new ternary shuttle vector is needed for plant virus infectious cloning.
Disclosure of Invention
In order to shorten the flow and time of gene operation and improve the research efficiency, the invention provides an escherichia coli-yeast-agrobacterium shuttle vector which can replicate and proliferate in escherichia coli, agrobacterium and yeast cells and can be used for carrying out efficient, rapid and high-flux DNA transformation to fungi or plant cells.
The technical scheme provided by the invention is as follows: an E.coli-yeast-Agrobacterium shuttle vector is a ternary vector containing a yeast DNA replicon and a yeast selection marker gene.
The invention provides an escherichia coli-yeast-agrobacterium ternary shuttle vector, which is formed by recombining yeast DNA replicons into an escherichia coli-agrobacterium binary shuttle vector, wherein the escherichia coli-agrobacterium binary shuttle vector is pCB301 (accession number is AF 139061.1) or pCass4-Rz; the pCB301 or pCass4-Rz contains a DNA replicon of escherichia coli, a DNA replicon pVS1-origin and a T-DNA sequence of agrobacterium and a Kana resistance gene; the yeast DNA replicon is pGBK; the sequence of the ternary shuttle vector is shown as SEQ ID No.1 or SEQ ID No. 2. The sequence of pCass4-Rz is shown as SEQ ID No. 3.
Further, the escherichia coli-yeast-agrobacterium ternary shuttle vector also contains a yeast screening marker gene.
Further, the yeast selection marker gene is TRP1.
Further, the yeast screening marker gene indicates whether the target gene carried by the escherichia coli-yeast-agrobacterium ternary shuttle vector is expressed in the receptor cell or not through an SD-Trp selective medium.
Further, a restriction enzyme SacII single-enzyme digestion binary vector plasmid pCB301 plasmid, pCass4-Rz plasma id is adopted to obtain a linearized vector.
Further, the enzyme digestion reaction system is as follows: plasmid pCB301 plasmid, pCass4-Rz plasma id 3. Mu.L, restriction enzyme Sac II 0.5. Mu.L, 10 XT Buffer 2. Mu.L, BSA 2. Mu.L, ddH 2 O12.5. Mu.L; cleavage reaction conditions: reacting for 12h at 37 ℃; and recovering the target linearization carrier by ethanol precipitation, and checking the correctness of the strip by electrophoresis.
Further, pGBKT7 plasmid is used as a template, PCR amplification is carried out by using primers PGBK2686-F1, PGBK2686-R1, PGBK2686-F2 and PGBK2686-R2 respectively, two DNA fragments of 2.6Kb containing a yeast replication element 2 mu sequence and Y1 and Y2 of a screening marker gene sequence are obtained, and the base sequences of the primers PGBK-2686F1 and PGBK-2686R1 are as follows:
forward primer PGBK2686-F1 (5 '. Fwdarw.3'): GCCGTGTGCGAGACACCGCGGCACATTTCCCCGAAAAGTGCCACC;
reverse primer PGBK2686-R1 (5 '. Fwdarw.3'): CCACAACGCCGGCGGCCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC;
forward primer PGBK2686-F2 (5 '. Fwdarw.3'): GTTGCTGCCTGTGATCACCGCGGCACATTTCCCCGAAAAGTGCCACC;
reverse primer PGBK2686-R2 (5 '. Fwdarw.3'): CGACGGAGCCGATTTTGAAACCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC.
The invention also provides application of the escherichia coli-yeast-agrobacterium shuttle vector in plant virus infectious cloning.
Further, the application includes:
(1) Transforming a target gene and the escherichia coli-yeast-agrobacterium shuttle vector into a yeast cell, and performing yeast homologous recombination to obtain a recombinant vector;
(2) Transferring the recombinant vector containing the target gene into fungi or plant cells by an agrobacterium-mediated transformation method, and successfully detecting the expression of the target gene.
Further, restriction enzymes Stu I and Bam I are adopted to cleave plasmid pCA4Y, and a vector linearized by pCA4Y is obtained; the genome of the full-length cDNA of the cucumber mosaic virus is amplified in a segmented way by taking the full-length cDNA of the cucumber mosaic virus as a template, and CMV-Fny/RNA1, RNA2, yeast/StuI/F, CMV-Fny/RNA1, yeast/BamHI/R, CMV-Fny/RNA2, yeast/BamHI/R, CMV-Fny/RNA3, yeast/StuI/F, CMV-Fny/RNA3, yeast/BamHI/R as primers to obtain CMV-RNA1, RNA2 and RNA3 fragments; the base sequences of the primers of each pair are as follows:
the forward primer CMV-Fny/RNA1& RNA 2/year/StuI/F (5 '. Fwdarw.3'): GTTCATTTCATTTGGAGAGGGTTTATTTACAAGAGCGTACGGTTCAATCC;
reverse primer CMV-Fny/RNA 1/year/BamHI/R (5 '. Fwdarw.3'): CATCCGGTGACAGGGTATCGTGGTCTCCTTTTAGAGACCCCCACGAAAG;
reverse primer CMV-Fny/RNA 2/year/BamHI/R (5 '. Fwdarw.3'): CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCACAAAAG;
the forward primer CMV-Fny/RNA3/yeast/StuI/F (5 '. Fwdarw.3'): GTTCATTTCATTTGGAGAGGGTAATCTTACCACTGTGTGTGTGCGTG (;
reverse primer CMV-Fny/RNA 3/year/BamHI/R (5 '. Fwdarw.3'): CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCCACG.
The invention recombines yeast DNA replicon into binary vector, the replication origin of yeast DNA replicon is identified by replication related protein of yeast cell, thus the E.coli-yeast-agrobacterium shuttle vector obtains the ability of replication, proliferation and stable existence in yeast cell for high-efficiency, rapid and high-flux DNA transformation.
Preferably, the binary vector is pCB301 or pCass-4Rz.
The pCB301 and pCass-4Rz plasmids contain DNA replicons of escherichia coli, pVS1_origin and T-DNA sequences of agrobacterium and Kana resistance genes, which are common screening genes of escherichia coli and agrobacterium, and can be directly used as screening markers without additionally inserting the resistance genes. Of course, the Kana resistance gene may be replaced with Amp, tet, cmr and Spec resistance genes as desired.
In the invention, pGBK is selected as the yeast DNA replicon, and the yeast DNA replicon has a2 mu sequence.
In order to facilitate the screening of successfully transformed colonies of the E.coli-yeast-Agrobacterium shuttle vector after transformation of the yeast cells, the E.coli-yeast-Agrobacterium shuttle expression vector also contains a yeast screening marker gene. Preferably, the yeast selection marker gene is TRP1, and the marker gene can be used for indicating whether the target gene carried by the escherichia coli-yeast-agrobacterium shuttle vector is expressed in a receptor cell through an SD-Trp selective medium.
The E.coli-yeast-Agrobacterium shuttle vector of the invention also contains a 2. Mu. Ori promoter sequence.
The construction method of the escherichia coli-yeast-agrobacterium shuttle vector comprises the following steps:
and (3) carrying out recombination reaction on the yeast DNA replicon, the yeast screening gene and the initial vector, converting the escherichia coli cells, and carrying out homologous recombination on the escherichia coli to obtain the escherichia coli-yeast-agrobacterium shuttle vector.
The invention also provides a recombinant strain containing the escherichia coli-yeast-agrobacterium shuttle vector. The recombinant strain is yeast, escherichia coli or agrobacterium.
The invention also provides application of the escherichia coli-yeast-agrobacterium shuttle vector in transforming DNA into plant cells. The shuttle vector can be used for realizing the rapid construction of a recombinant vector containing a target gene, the rapid transformation in fungi or plant cells and the efficient screening of mutants.
Specifically, the application includes:
(1) Transforming a target gene and the escherichia coli-yeast-agrobacterium shuttle vector into a yeast cell, obtaining a recombinant expression vector through yeast homologous recombination, and picking a transformant on a selective medium;
(2) The recombinant expression vector is transferred into plant cells by an agrobacterium-mediated transformation method.
When used for gene knockout, the upstream homologous sequence and the downstream homologous sequence of the gene to be knocked out are cloned from the genome of the receptor cell, and the upstream homologous sequence and the downstream homologous sequence, the proper resistance gene and the digested escherichia coli-yeast-agrobacterium shuttle vector are mixed to transform the yeast competent cells; obtaining a gene knockout vector containing a gene knockout structure (an upstream homologous sequence of a gene to be knocked out-a resistance gene-a downstream homologous sequence of the gene to be knocked out) through yeast homologous recombination; the gene knockout vector is transformed into agrobacterium, and then transformed into receptor cells by an ATMT method, and transformants are picked up on a selective medium.
When used for gene expression, the gene of interest and its promoter are cloned, or only the coding region of the gene of interest is cloned, and in addition, the appropriate promoters from other sources are cloned by PCR; then mixing target genes with promoters or coding regions of the target genes with proper promoters, proper resistance genes and enzyme-cut escherichia coli-yeast-agrobacterium shuttle vectors to transform yeast competent cells; obtaining a recombinant vector containing a target gene through yeast homologous recombination; transforming agrobacterium with the recombinant vector, picking the transformant on a selective culture medium, and transforming the transformant into a receptor cell by an ATMT method; observing the expression symptoms of the target gene in the plant, extracting the total protein of the transformed cells (plants or fungi), and identifying whether the target gene is expressed and the expression quantity thereof by adopting methods such as Western blot or enzyme activity measurement and the like according to the characteristics of the target protein.
The E.coli-yeast-Agrobacterium shuttle vector of the invention can be used for transforming DNA into fungi or plant cells, but has very low DNA recombination efficiency due to the specificity of plant cells, so that the E.coli-yeast-Agrobacterium shuttle vector can only be used for realizing gene expression in plant cells and can not be used for realizing gene knockout in plant cells.
Compared with the prior art, the invention has the beneficial effects that:
the escherichia coli-yeast-agrobacterium shuttle vector disclosed by the invention is a plasmid which can be copied, proliferated and stably exist in yeast, escherichia coli and agrobacterium at the same time, and can be used for directly transforming fungi or plant cells in an agrobacterium-mediated mode, so that the flow and time of gene operation are shortened, the use is convenient, the working efficiency is high, and the preparation process is simple, convenient, rapid and efficient. The correlation of viruses with hosts is often an important topic of research in the virology field, and the most advantageous research tool in this field is the construction of virus-invasive clones. When the escherichia coli is used for constructing the full-length cDNA invasive clone of the large genome RNA virus, the coded viral protein can have toxic effect on host bacteria, so that nonspecific recombination is caused, and an unstable phenomenon occurs. In-vivo homologous recombination is an assembly method for realizing a plurality of DNA fragments with homologous sequences mutually existing by utilizing a high-efficiency homologous recombination system in yeast cells. The invention can overcome the instability of virus genome sequence cloning in E.coli, and establishes a yeast recombinant cloning system by constructing a ternary shuttle vector and then utilizing virus invasive cloning, thereby having the advantages of rapidness, convenience and low cost.
In addition, the present invention provides pCA4Y&The application of the two innovative vectors pCB3Y in the construction of virus infectious clones, such as the construction of infectious clones of cucumber mosaic virus; if the traditional plasmid is used, the plasmid carrying CMV RNA2 cDNA can not be obtained, and the plasmid has toxicity to colibacillus, so that the transformed bacterium with the correct plasmid can not survive, and the colibacillus and virus are similar in coding mode, polycistronic and microzyme is eukaryotic and monocistronic, so that the expression of toxic gene on virus genome can be avoided, and the stable pCA4Y-RNA2 capable of avoiding toxicity can be obtained by adopting the method CMV OR pCB3Y-RNA2 CMV The method comprises the steps of carrying out a first treatment on the surface of the The traditional analytical cloning is carried out in vitro by an enzyme digestion or recombination method, the adopted enzyme has higher cost, and the recombination in the experiment is carried out by the self-contained recombinase in the yeast body, so that the cost is lower and the efficiency is higher compared with the in vitro recombination mode; in addition, the yeast recombinant construction plasmid also has the advantage of large fragment recombination.
In a plant virus invasive cloning framework, the ternary shuttle vector can utilize saccharomycetes to carry out multi-fragment recombination to obtain plant virus invasive clones which possibly have toxicity in a procaryon and cannot be constructed, and the obtained clones can directly transform fungi or plant cells in an agrobacterium-mediated mode. In the construction of plant virus infectious clone, the ternary shuttle vector obviously shortens the flow and time of gene operation, is convenient to use and high in working efficiency, and avoids the situation that positive transformants cannot be obtained in escherichia coli due to high toxicity of virus proteins, and the preparation process is simple, convenient, rapid and efficient.
Drawings
FIG. 1 is a plasmid map of pCB301 vector;
FIG. 2 is a plasmid map of pCB3Y vector;
FIG. 3 is a plasmid map of the pCass-4Rz vector;
FIG. 4 is a plasmid map of the pCA4Y vector;
FIG. 5 is a plasmid map of pGBKT7 vector;
FIG. 6 is a diagram showing the result of electrophoresis of PCR amplified target gene using the transformant after recombination of the target gene with the ternary shuttle plasmid;
FIG. 7 is a graph showing tobacco infection results with a transformant after recombination of a viral gene of interest with a ternary shuttle plasmid;
FIG. 8 is a graph showing the result of PCR verification of RNA extracted from tobacco with toxicity.
Detailed Description
The construction method of the escherichia coli-yeast-agrobacterium shuttle vector pCB3Y or pCA4Y comprises the following steps:
obtaining pCB301 and pCass-4Rz escherichia coli-agrobacterium shuttle vectors;
construction of E.coli-Yeast-Agrobacterium shuttle vector:
the restriction enzyme Sac II single enzyme is adopted to cut the binary vector plasmid pCB301 plasmid, pCass-Rz plasma id, thus obtaining the linearized vector. Enzyme digestion reaction system: plasmid pCB301 plasmid, pCass4-Rz plasma id 3. Mu.L, restriction enzyme Sac II 0.5. Mu.L, 10 XT Buffer 2. Mu.L, BSA 2. Mu.L, ddH 2 O12.5. Mu.L. Cleavage reaction conditions: reacting for 12h at 37 ℃; and recovering the target linearization carrier by ethanol precipitation, and checking the correctness of the strip by electrophoresis.
PCR amplification is carried out by taking pGBK plasmid as a template and using primers pGBK2686-F1/pGBK2686-R1 and pGBK2686-F2/pGBK2686-R2 to obtain two DNA fragments of 2.6Kb yeast DNA replicon Y1 and Y2, wherein the base sequences of the primers PGBK-2686F1 and PGBK-2686R1 are as follows:
forward primer (SEQ ID No. 4) PGBK2686-F 1 (5′→3′)：CCGCGGCACATTTCCCCGAAAAGTGCCACC (SacII cleavage site underlined);
reverse primer (SEQ ID No. 5) PGBK2686-R 1 (5′→3′)：CCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC (SacII cleavage site underlined);
forward primer (SEQ ID No. 6) PGBK2686-F 2 (5′→3′)：CCGCGGCACATTTCCCCGAAAAGTGCCACC (SacII cleavage site underlined);
reverse primer (SEQ ID No. 7) PGBK2686-R 2 (5′→3′)：CCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC (SacII cleavage site underlined);
the PCR reaction system is as follows: template plasmid pGBK 1. Mu.L, phanta Max Super-Fidelity DNA polymerase. Mu.L, dNTP Mix (10 mM each) 1. Mu.L, 2x phanta Max Buffer 25. Mu.L, forward and reverse primers 2.5. Mu.L each, and water to 50. Mu.L.
The PCR reaction conditions for the yeast DNA replicon Y1 (SEQ ID No. 8) were: 94℃for 3 minutes, 28 cycles (94℃for 30 seconds, 60℃for 30 seconds, 72℃for 2 minutes and 50 seconds) and 72℃for 10 minutes.
The PCR reaction conditions for the yeast DNA replicon Y2 (SEQ ID No. 9) were: 94℃for 3 minutes, 28 cycles (94℃for 30 seconds, 60℃for 30 seconds, 72℃for 2 minutes and 50 seconds) and 72℃for 10 minutes.
And after the PCR product is verified to be correct by electrophoresis, recovering the target fragment by using a DNA gel purification kit.
Recombination reaction system: linearized pCB301 plasmid, pCass4-Rz plasma id vector plus 3. Mu.L, insert Y1, Y2 plus 0.5. Mu.L, 5 XCE buffer 4. Mu.L, T4 ligase 2. Mu.L, ddH, respectively 2 O10.5. Mu.L total 20. Mu.L system;
the recombinant plasmid is transformed into competent cell JM 110 of Escherichia coli, colonies growing on kanamycin LB plates are selected for culture, plasmids are extracted, PCR experiments are carried out to verify whether the recombinant vector contains 2 mu sequences of yeast replication elements and screening marker gene sequences, and the sequences are sent to a company for sequencing. And (3) cutting pCB3Y or pCA4Y by using Hind III enzyme, recovering the cut small fragment glue, sequencing by using pGBK2686F1 and pGBK2686F2, sequencing to obtain sequences, and respectively comparing the sequences with Y1 and Y2, wherein the sequences are positive. The vectors that were verified to be correct were designated as pCB3Y (as shown in fig. 2) or pCA4Y (as shown in fig. 4).
Construction of CMV expression vectors:
plasmid pCA4Y is digested by restriction enzymes Stu I and Bam I to obtain pCA4Y linearized vector. Enzyme digestion reaction system: plasmid pCA4Y 20. Mu.L, restriction enzymes Stu I and Bam I1. Mu.L each, 10 XK Buffer 5. Mu.L, ddH 2 O23. Mu.L. Cleavage reaction conditions: enzyme digestion reaction is carried out for 10 hours at 30 ℃; and recovering the target linearization carrier by ethanol precipitation, and checking the correctness of the strip by electrophoresis.
The genome of the full-length cDNA of cucumber mosaic virus (Cucumber mosaic virus, CMV) is amplified in a segmented way by taking the full-length cDNA of the cucumber mosaic virus (Cucumber mosaic virus, CMV) as a template, namely CMV-Fny/RNA1& RNA2/yeast/StuI/F, CMV-Fny/RNA1/yeast/BamHI/R, CMV-Fny/RNA2/yeast/BamHI/R, CMV-Fny/RNA3/yeast/StuI/F, CMV-Fny/RNA3/yeast/BamHI/R as a primer to obtain CMV-RNA1/RNA2/RNA3 fragments. The base sequences of the primers of each pair are as follows:
forward primer CMV-Fny/RNA1&RNA2/yeast/StuI/F(5′→3′)：GTTCATTTCATTTGGAGAGGGTTTATTTACAAGAGCGTACGGTTCAATCC (underlined is the sequence homologous to the pCA4Y vector);
reverse primer CMV-Fny/RNA 1/year/BamHI/R (5 '. Fwdarw.3'):CATCCGGTGACAGGGTATCGTGGTCTCCTTTTAGAGACCCCCACGAAAG (underlined is the sequence homologous to the pCA4Y vector);
reverse primer CMV-Fny/RNA 2/year/BamHI/R (5 '. Fwdarw.3'):CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCACAAAAG (underlined is a sequence homologous to the pCAY vector);
the forward primer CMV-Fny/RNA3/yeast/StuI/F (5 '. Fwdarw.3'):GTTCATTTCATTTGGAGAGGGTAATCTTACCACTGTGTGTGTGCGTG (underlined is the sequence homologous to the pCA4Y vector);
reverse primer CMV-Fny/RNA 3/year/BamHI/R (5 '. Fwdarw.3'):CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCCACG (underlined is the sequence homologous to the pCA4Y vector);
the PCR reaction system is as follows: template plasmid 1. Mu.L, phanta Max Super-Fidelity DNA polymerase 0.5.5. Mu.L, dNTP Mix (10 mM each) 0.5. Mu.L, 2x phanta Max Buffer 10. Mu.L, forward and reverse primers 0.25. Mu.L each, and water to 20. Mu.L.
The PCR reaction conditions of CMV-Fny/RNA1 were: 95℃for 3min,95℃for 15s,63℃for 15s,72℃for 3min for 40s,28 cycles, 72℃for 5min,10℃for 1min,4℃for preservation;
the PCR reaction conditions of CMV-Fny/RNA2 were: 3min at 95 ℃, 15s at 65 ℃, 3min at 72 ℃,10 s at 28 cycles, 5min at 72 ℃, 1min at 10 ℃,4 ℃ for preservation;
the PCR reaction conditions of CMV-Fny/RNA3 were: 95℃for 3min,95℃for 15s,67℃for 15s,72℃for 2min for 30s,28 cycles, 72℃for 5min,10℃for 1min,4 ℃.
And after the PCR product is verified to be correct by electrophoresis, recovering the target fragment by using a DNA gel purification kit.
Preparation of Yeast competence:
culturing activated YPH500 in 2xYPDA culture medium in 30 ℃ incubator for one to two days, selecting bacteria to test tube containing 4mL 2xYPDA solution and culturing overnight in 30 ℃ incubator; sucking 2-3 ml of bacterial liquid in a test tube into 100ml of 2xYPDA solution and shake culturing at 30 ℃ for 5-6 hours until turbidity; transferring the bacterial liquid into a 50mL centrifuge tube, centrifuging at 400xg for 5min, discarding the supernatant, re-suspending the cells with 9mL TE (pH 7.5), centrifuging, and discarding the supernatant; 5mL of Lithoium/Concessium Acetate was resuspended and then gently shaken at 30℃for 30 minutes on a 45-turn shaker; centrifuging at 400xg for 5min, and discarding the supernatant; competent cells were obtained by resuspension with 1mL TE (pH 7.5). ,
transformation of Yeast competent cells:
taking 100 mu L to 1.5mL of prepared yeast competent cells into a centrifuge tube, and sequentially adding the following reagents: histamine solution 5. Mu.L, carrier DNA 5. Mu.L and pCA4Y linearized vector with 500ng each of CMV-RNA1/RNA2/RNA3 fragments; gently mixing, and incubating at room temperature for 15min. The mixture was added to the system after being mixed well in another centrifuge tube, 0.8mL PEG,0.2mL TE/introduction miax. Incubating at 30 ℃ for 10min, connecting with a connector at 42 ℃ for 30min, incubating at 30 ℃ for 5min, centrifuging at 700xg for 5-10 seconds, discarding supernatant, adding 200 mu L of non-anti-SOC solution for resuspension, uniformly coating on a Trp defect type screening plate, and culturing at 30 ℃ for 2-4 d.
Screening positive yeast cells on the plates, extracting yeast plasmids by using a yeast plasmid extraction kit, transforming the plasmids into competent cells XL10 of escherichia coli, picking positive colonies growing on kanamycin LB plates for culture, extracting plasmids, and identifying the plasmids by PCR (primers are pGBK2686-F2/pGBK2686-R2, CMV-Fny/RNA1& RNA2/yeast/StuI/F, CMV-Fny/RNA1/yeast/BamHI/R, CMV-Fny/RNA2/yeast/BamHI/R, CMV-Fny/RNA3/yeast/StuI/F, CMV-Fny/RNA3/yeast/BamHI/R respectively). The obtained CMV cDNA infectious clone vectors which verify correctness are named pCA4Y-CMV/RNA1 (SEQ ID No. 10), pCA4Y-CMV/RNA2 (SEQ ID No. 11) and pCA4Y-CMV/RNA3 (SEQ ID No. 12), respectively.
Plasmid 1: pCB3Y-RNA1 CMV
CMV-Fny/RNA1&RNA2/yeast/StuI/F:GTTCATTTCATTTGGAGAGGGTTTATTTACAAGAGCGTACGGTTCAATCC
CMV-Fny/RNA1/yeast/BamHI/R:CATCCGGTGACAGGGTATCGTGGTCTCCTTTTAGAGACCCCCACGAAAG
Plasmid 2: pCB3Y-RNA2 CMV
CMV-Fny/RNA1&RNA2/yeast/StuI/F:GTTCATTTCATTTGGAGAGGGTTTATTTACAAGAGCGTACGGTTCAATCC
CMV-Fny/RNA2/yeast/BamHI/R:CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCACAAAAG
Plasmid 3: pCB3Y-RNA2 CMV
CMV-Fny/RNA3/yeast/StuI/F:GTTCATTTCATTTGGAGAGGGTAATCTTACCACTGTGTGTGTGCGTG
CMV-Fny/RNA3/yeast/BamHI/R:CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCCACG
The vectors pCA4Y-CMV/RNA1, pCA4Y-CMV/RNA2 and pCA4Y-CMV/RNA3 are transformed into the agrobacterium strain GV3101 by a freeze thawing method and inoculated with the present smoke, and the specific steps are as follows:
the agrobacterium solution identified as positive clone was added to LB liquid medium containing Kana and Rif antibiotics, cultured at 28℃for 12-16 h with shaking at 200 r/min. The cells were collected by centrifugation and suspended in an Agrobacterium buffer to an OD600 of 0.3. After standing for 2 hours, the smoke was inoculated with a syringe, and three repeated experimental groups were set. Healthy raw tobacco is used as a negative control; positive agrobacterium with CMV genome was used as positive control. Tobacco symptoms were observed 7-10 d after inoculation and RNA was extracted and assayed for infectivity by RT-PCR.
PCB3Y vector sequence (SEQ ID No. 1)
aagcttgcatgcctgcagtcaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccagagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaagatggcttctacaaatgccatcattgcgataaaggaaaggctatcgttcaaagaatgcctctaccgacagtggtcccaaagatggaccccccacccacgaggaacatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgataacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccagagggctattgagactttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaagatggcttctacaaatgccatcattgcgataaaggaaaggctatcgttcaagaatgcctctaccgacagtggtcccaaagatggacccccacccacgaggaacatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggcctgacctgcaggtcgactctagaggatccccgggtcggcatggcatctccacctcctcgcggtccgacctgggcatccgaaggaggacgtcgtccactcggatggctaagggagagctcgaatttccccgatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcggaattcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataatcggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacaggagatctcagtaaagcgctggctgaacccccagccggaactgaccccacaaggccctagcgtttgcaatgcaccaggtcatcattgacccaggcgtgttccaccaggccgctgcctcgcaactcttcgcaggcttcgccgacctgctcgcgccacttcttcacgcgggtggaatccgatccgcacatgaggcggaaggtttccagcttgagcgggtacggctcccggtgcgagctgaaatagtcgaacatccgtcgggccgtcggcgacagcttgcggtacttctcccatatgaatttcgtgtagtggtcgccagcaaacagcacgacgatttcctcgtcgatcaggacctggcaacgggacgttttcttgccacggtccaggacgcggaagcggtgcagcagcgacaccgattccaggtgcccaacgcggtcggacgtgaagcccatcgccgtcgcctgtaggcgcgacaggcattcctcggccttcgtgtaataccggccattgatcgaccagcccaggtcctggcaaagctcgtagaacgtgaaggtgatcggctcgccgataggggtgcgcttcgcgtactccaacacctgctgccacaccagttcgtcatcgtcggcccgcagctcgacgccggtgtaggtgatcttcacgtccttgttgacgtggaaaatgaccttgttttgcagcgcctcgcgcgggattttcttgttgcgcgtggtgaacagggcagagcgggccgtgtcgtttggcatcgctcgcatcgtgtccggccacggcgcaatatcgaacaaggaaagctgcatttccttgatctgctgcttcgtgtgtttcagcaacgcggcctgcttggcctcgctgacctgttttgccaggtcctcgccggcggtttttcgcttcttggtcgtcatagttcctcgcgtgtcgatggtcatcgacttcgccaaacctgccgcctcctgttcgagacgacgcgaacgctccacggcggccgatggcgcgggcagggcagggggagccagttgcacgctgtcgcgctcgatcttggccgtagcttgctggaccatcgagccgacggactggaaggtttcgcggggcgcacgcatgacggtgcggcttgcgatggtttcggcatcctcggcggaaaaccccgcgtcgatcagttcttgcctgtatgccttccggtcaaacgtccgattcattcaccctccttgcgggattgccccgactcacgccggggcaatgtgcccttattcctgatttgacccgcctggtgccttggtgtccagataatccaccttatcggcaatgaagtcggtcccgtagaccgtctggccgtccttctcgtacttggtattccgaatcttgccctgcacgaataccagcgaccccttgcccaaatacttgccgtgggcctcggcctgagagccaaaacacttgatgcggaagaagtcggtgcgctcctgcttgtcgccggcatcgttgcgccacatctaggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcgatcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccacaattatgggtgatgctgccaactcgagagcgggccgggagggttcgagaagggggggcaccccccttcggcgtgcgcggtcacgcgcacagggcgcagccctggttaaaaacaaggtttataaatattggtttaaaagcaggttaaaagacaggttagcggtggccgaaaaacgggcggaaacccttgcaaatgctggattttctgcctgtggacagcccctcaaatgtcaataggtgcgcccctcatctgtcagcactctgcccctcaagtgtcaaggatcgcgcccctcatctgtcagtagtcgcgcccctcaagtgtcaataccgcagggcacttatccccaggcttgtccacatcatctgtgggaaactcgcgtaaaatcaggcgttttcgccgatttgcgaggctggccagctccacgtcgccggccgaaatcgagcctgcccctcatctgtcaacgccgcgccgggtgagtcggcccctcaagtgtcaacgtccgcccctcatctgtcagtgagggccaagttttccgcgaggtatccacaacgccggcggccgcggcacatttccccgaaaagtgccacctgaacgaagcatctgtgcttcattttgtagaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgaaagcgctattttaccaacgaagaatctgtgcttcatttttgtaaaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgagagcgctattttaccaacaaagaatctatacttcttttttgttctacaaaaatgcatcccgagagcgctatttttctaacaaagcatcttagattactttttttctcctttgtgcgctctataatgcagtctcttgataactttttgcactgtaggtccgttaaggttagaagaaggctactttggtgtctattttctcttccataaaaaaagcctgactccacttcccgcgtttactgattactagcgaagctgcgggtgcattttttcaagataaaggcatccccgattatattctataccgatgtggattgcgcatactttgtgaacagaaagtgatagcgttgatgattcttcattggtcagaaaattatgaacggtttcttctattttgtctctatatactacgtataggaaatgtttacattttcgtattgttttcgattcactctatgaatagttcttactacaatttttttgtctaaagagtaatactagagataaacataaaaaatgtagaggtcgagtttagatgcaagttcaaggagcgaaaggtggatgggtaggttatatagggatatagcacagagatatatagcaaagagatacttttgagcaatgtttgtggaagcggtattcgcaatattttagtagctcgttacagtccggtgcgtttttggttttttgaaagtgcgtcttcagagcgcttttggttttcaaaagcgctctgaagttcctatactttctagagaataggaacttcggaataggaacttcaaagcgtttccgaaaacgagcgcttccgaaaatgcaacgcgagctgcgcacatacagctcactgttcacgtcgcacctatatctgcgtgttgcctgtatatatatatacatgagaagaacggcatagtgcgtgtttatgcttaaatgcgtacttatatgcgtctatttatgtaggatgaaaggtagtctagtacctcctgtgatattatcccattccatgcggggtatcgtatgcttccttcagcactaccctttagctgttctatatgctgccactcctcaattggattagtctcatccttcaatgctatcatttcctttgatattggatcatactaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatagatcaacgacattactatatatataatataggaagcatttaatagaacagcatcgtaatatatgtgtactttgcagttatgacgccagatggcagtagtggaagatattctttattgaaaaatagcttgtcaccttacgtacaatcttgatccggagcttttctttttttgccgattaagaattaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatgtctgttattaatttcacaggtagttctggtccattggtgaaagtttgcggcttgcagagcacagaggccgcagaatgtgctctagattccgatgctgacttgctgggtattatatgtgtgcccaatagaaagagaacaattgacccggttattgcaaggaaaatttcaagtcttgtaaaagcatataaaaatagttcaggcactccgaaatacttggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggcaagaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagcttcacagaaacctcattcgtttattcccttgtttgattcagaagcaggtgggacaggtgaacttttggattggaactcgatttctgactgggttggaaggcaagagagccccgaaagcttacattttatgttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaagactctaacaaaatagcaaatttcgtcaaaaatgctaagaaataggttattactgagtagtatttatttaagtattgtttgtgcacttgccgatctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggccgcggtgtctcgcacacggcttcgacggcgtttctggcgcgtttgcagggccatagacggccgccagcccagcggcgagggcaaccagcccggtgagcgtctagtggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactggggtggtttt
PCA4Y vector sequence (SEQ ID No. 2)
gaattctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccagagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaagatggcttctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctaccgacagtggtcccaaagatggacccccacccacgaggaacatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatggtcaacatggtggagcacgacactctcgtctactccaagaatatcaaagatacagtctcagaagaccagagggctattgagacttttcaacaaagggtaatatcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaagatggcttctacaaatgccatcattgcgataaaggaaaggctatcgttcaagatgcctctaccgacagtggtcccaaagatggacccccacccacgaggaacatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggcctgggtacctctagaggatccgataccctgtcaccggatgtgttttccggtctgatgagtccgtgaggacgaaacaggactgtcctgcaggagctcgaattcggtacgctgaaatcaccagtctctctctacaaatctatctctctctattttctccataaataatgtgtgagtagtttccgataaggaaattagggttcttatagggtttcgtcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatcagtactaaatcagatccccaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagtggcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgctagagcagcttgagcttggatcagattgtcgtttcccgccttcagtttaaactatcagtgtttgacaggatatattggcgggtaaacctaagagaaaagagcgtttattagaataacggatatttaaaagggcgtgaaaaggtttatccgttcgtccatttgtatgtgcatgccaaccacagggttcccctcgggatcaaagtactttgatccaacccctccgctgctatagtgcagtcggcttctgacgttcagtgcagccgtcttctgaaaacgacatgtcgcacaagtcctaagttacgcgacaggctgccgccctgcccttttcctggcgttttcttgtcgcgtgttttagtcgcataaagtagaatacttgcgactagaaccggagacattacgccatgaacaagagcgccgccgctggcctgctgggctatgcccgcgtcagcaccgacgaccaggacttgaccaaccaacgggccgaactgcacgcggccggctgcaccaagctgttttccgagaagatcaccggcaccaggcgcgaccgcccggagctggccaggatgcttgaccacctacgccctggcgacgttgtgacagtgaccaggctagaccgcctggcccgcagcacccgcgacctactggacattgccgagcgcatccaggaggccggcgcgggcctgcgtagcctggcagagccgtgggccgacaccaccacgccggccggccgcatggtgttgaccgtgttcgccggcattgccgagttcgagcgttccctaatcatcgaccgcacccggagcgggcgcgaggccgccaaggcccgaggcgtgaagtttggcccccgccctaccctcaccccggcacagatcgcgcacgcccgcgagctgatcgaccaggaaggccgcaccgtgaaagaggcggctgcactgcttggcgtgcatcgctcgaccctgtaccgcgcacttgagcgcagcgaggaagtgacgcccaccgaggccaggcggcgcggtgccttccgtgaggacgcattgaccgaggccgacgccctggcggccgccgagaatgaacgccaagaggaacaagcatgaaaccgcaccaggacggccaggacgaaccgtttttcattaccgaagagatcgaggcggagatgatcgcggccgggtacgtgttcgagccgcccgcgcacgtctcaaccgtgcggctgcatgaaatcctggccggtttgtctgatgccaagctggcggcctggccggccagcttggccgctgaagaaaccgagcgccgccgtctaaaaaggtgatgtgtatttgagtaaaacagcttgcgtcatgcggtcgctgcgtatatgatgcgatgagtaaataaacaaatacgcaaggggaacgcatgaaggttatcgctgtacttaaccagaaaggcgggtcaggcaagacgaccatcgcaacccatctagcccgcgccctgcaactcgccggggccgatgttctgttagtcgattccgatccccagggcagtgcccgcgattgggcggccgtgcgggaagatcaaccgctaaccgttgtcggcatcgaccgcccgacgattgaccgcgacgtgaaggccatcggccggcgcgacttcgtagtgatcgacggagcgccccaggcggcggacttggctgtgtccgcgatcaaggcagccgacttcgtgctgattccggtgcagccaagcccttacgacatatgggccaccgccgacctggtggagctggttaagcagcgcattgaggtcacggatggaaggctacaagcggcctttgtcgtgtcgcgggcgatcaaaggcacgcgcatcggcggtgaggttgccgaggcgctggccgggtacgagctgcccattcttgagtcccgtatcacgcagcgcgtgagctacccaggcactgccgccgccggcacaaccgttcttgaatcagaacccgagggcgacgctgcccgcgaggtccaggcgctggccgctgaaattaaatcaaaactcatttgagttaatgaggtaaagagaaaatgagcaaaagcacaaacacgctaagtgccggccgtccgagcgcacgcagcagcaaggctgcaacgttggccagcctggcagacacgccagccatgaagcgggtcaactttcagttgccggcggaggatcacaccaagctgaagatgtacgcggtacgccaaggcaagaccattaccgagctgctatctgaatacatcgcgcagctaccagagtaaatgagcaaatgaataaatgagtagatgaattttagcggctaaaggaggcggcatggaaaatcaagaacaaccaggcaccgacgccgtggaatgccccatgtgtggaggaacgggcggttggccaggcgtaagcggctgggttgtctgccggccctgcaatggcactggaacccccaagcccgaggaatcggcgtgacggtcgcaaaccatccggcccggtacaaatcggcgcggcgctgggtgatgacctggtggagaagttgaaggccgcgcaggccgcccagcggcaacgcatcgaggcagaagcacgccccggtgaatcgtggcaagcggccgctgatcgaatccgcaaagaatcccggcaaccgccggcagccggtgcgccgtcgattaggaagccgcccaagggcgacgagcaaccagattttttcgttccgatgctctatgacgtgggcacccgcgatagtcgcagcatcatggacgtggccgttttccgtctgtcgaagcgtgaccgacgagctggcgaggtgatccgctacgagcttccagacgggcacgtagaggtttccgcagggccggccggcatggccagtgtgtgggattacgacctggtactgatggcggtttcccatctaaccgaatccatgaaccgataccgggaagggaagggagacaagcccggccgcgtgttccgtccacacgttgcggacgtactcaagttctgccggcgagccgatggcggaaagcagaaagacgacctggtagaaacctgcattcggttaaacaccacgcacgttgccatgcagcgtacgaagaaggccaagaacggccgcctggtgacggtatccgagggtgaagccttgattagccgctacaagatcgtaaagagcgaaaccgggcggccggagtacatcgagatcgagctagctgattggatgtaccgcgagatcacagaaggcaagaacccggacgtgctgacggttcaccccgattactttttgatcgatcccggcatcggccgttttctctaccgcctggcacgccgcgccgcaggcaaggcagaagccagatggttgttcaagacgatctacgaacgcagtggcagcgccggagagttcaagaagttctgtttcaccgtgcgcaagctgatcgggtcaaatgacctgccggagtacgatttgaaggaggaggcggggcaggctggcccgatcctagtcatgcgctaccgcaacctgatcgagggcgaagcatccgccggttcctaatgtacggagcagatgctagggcaaattgccctagcaggggaaaaaggtcgaaaaggtctctttcctgtggatagcacgtacattgggaacccaaagccgtacattgggaaccggaacccgtacattgggaacccaaagccgtacattgggaaccggtcacacatgtaagtgactgatataaaagagaaaaaaggcgatttttccgcctaaaactctttaaaacttattaaaactcttaaaacccgcctggcctgtgcataactgtctggccagcgcacagccgaagagctgcaaaaagcgcctacccttcggtcgctgcgctccctacgccccgccgcttcgcgtcggcctatcgcggccgctggccgctcaaaaatggctggcctacggccaggcaatctaccagggcgcggacaagccgcgccgtcgccactcgaccgccggcgcccacatcaaggcaccctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgcattctaggtactaaaacaattcatccagtaaaatataatattttattttctcccaatcaggcttgatccccagtaagtcaaaaaatagctcgacatactgttcttccccgatatcctccctgatcgaccggacgcagaaggcaatgtcataccacttgtccgccctgccgcttctcccaagatcaataaagccacttactttgccatctttcacaaagatgttgctgtctcccaggtcgccgtgggaaaagacaagttcctcttcgggcttttccgtctttaaaaaatcatacagctcgcgcggatctttaaatggagtgtcttcttcccagttttcgcaatccacatcggccagatcgttattcagtaagtaatccaattcggctaagcggctgtctaagctattcgtatagggacaatccgatatgtcgatggagtgaaagagcctgatgcactccgcatacagctcgataatcttttcagggctttgttcatcttcatactcttccgagcaaaggacgccatcggcctcactcatgagcagattgctccagccatcatgccgttcaaagtgcaggacctttggaacaggcagctttccttccagccatagcatcatgtccttttcccgttccacatcataggtggtccctttataccggctgtccgtcatttttaaatataggttttcattttctcccaccagcttatataccttagcaggagacattccttccgtatcttttacgcagcggtatttttcgatcagttttttcaattccggtgatattctcattttagccatttattatttccttcctcttttctacagtatttaaagataccccaagaagctaattataacaagacgaactccaattcactgttccttgcattctaaaaccttaaataccagaaaacagctttttcaaagttgttttcaaagttggcgtataacatagtatcgacggagccgattttgaaaccgcggcacatttccccgaaaagtgccacctgaacgaagcatctgtgcttcattttgtagaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgaaagcgctattttaccaacgaagaatctgtgcttcatttttgtaaaacaaaaatgcaacgcgagagcgctaatttttcaaacaaagaatctgagctgcatttttacagaacagaaatgcaacgcgagagcgctattttaccaacaaagaatctatacttcttttttgttctacaaaaatgcatcccgagagcgctatttttctaacaaagcatcttagattactttttttctcctttgtgcgctctataatgcagtctcttgataactttttgcactgtaggtccgttaaggttagaagaaggctactttggtgtctattttctcttccataaaaaaagcctgactccacttcccgcgtttactgattactagcgaagctgcgggtgcattttttcaagataaaggcatccccgattatattctataccgatgtggattgcgcatactttgtgaacagaaagtgatagcgttgatgattcttcattggtcagaaaattatgaacggtttcttctattttgtctctatatactacgtataggaaatgtttacattttcgtattgttttcgattcactctatgaatagttcttactacaatttttttgtctaaagagtaatactagagataaacataaaaaatgtagaggtcgagtttagatgcaagttcaaggagcgaaaggtggatgggtaggttatatagggatatagcacagagatatatagcaaagagatacttttgagcaatgtttgtggaagcggtattcgcaatattttagtagctcgttacagtccggtgcgtttttggttttttgaaagtgcgtcttcagagcgcttttggttttcaaaagcgctctgaagttcctatactttctagagaataggaacttcggaataggaacttcaaagcgtttccgaaaacgagcgcttccgaaaatgcaacgcgagctgcgcacatacagctcactgttcacgtcgcacctatatctgcgtgttgcctgtatatatatatacatgagaagaacggcatagtgcgtgtttatgcttaaatgcgtacttatatgcgtctatttatgtaggatgaaaggtagtctagtacctcctgtgatattatcccattccatgcggggtatcgtatgcttccttcagcactaccctttagctgttctatatgctgccactcctcaattggattagtctcatccttcaatgctatcatttcctttgatattggatcatactaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatagatcaacgacattactatatatataatataggaagcatttaatagaacagcatcgtaatatatgtgtactttgcagttatgacgccagatggcagtagtggaagatattctttattgaaaaatagcttgtcaccttacgtacaatcttgatccggagcttttctttttttgccgattaagaattaattcggtcgaaaaaagaaaaggagagggccaagagggagggcattggtgactattgagcacgtgagtatacgtgattaagcacacaaaggcagcttggagtatgtctgttattaatttcacaggtagttctggtccattggtgaaagtttgcggcttgcagagcacagaggccgcagaatgtgctctagattccgatgctgacttgctgggtattatatgtgtgcccaatagaaagagaacaattgacccggttattgcaaggaaaatttcaagtcttgtaaaagcatataaaaatagttcaggcactccgaaatacttggttggcgtgtttcgtaatcaacctaaggaggatgttttggctctggtcaatgattacggcattgatatcgtccaactgcatggagatgagtcgtggcaagaataccaagagttcctcggtttgccagttattaaaagactcgtatttccaaaagactgcaacatactactcagtgcagcttcacagaaacctcattcgtttattcccttgtttgattcagaagcaggtgggacaggtgaacttttggattggaactcgatttctgactgggttggaaggcaagagagccccgaaagcttacattttatgttagctggtggactgacgccagaaaatgttggtgatgcgcttagattaaatggcgttattggtgttgatgtaagcggaggtgtggagacaaatggtgtaaaagactctaacaaaatagcaaatttcgtcaaaaatgctaagaaataggttattactgagtagtatttatttaagtattgtttgtgcacttgccgatctatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggccgcggtgatcacaggcagcaacgctctgtcatcgttacaatcaacatgctaccctccgcgagatcatccgtgtttcaaacccggcagcttagttgccgttcttccgaatagcatcggtaacatgagcaaagtctgccgccttacaacggctctcccgctgacgccgtcccggactgatgggctgcctgtatcgagtggtgattttgtgccgagctgccggtcggggagctgttggctggctggtggcaggatatattgtggtgtaaacaaattgacgcttagacaacttaataacacattgcggacgtttttaatgtactgaattaacgccgaattaattcgggggatctggattttagtactggattttggttttaggaattagaaattttattgatagaagtattttacaaatacaaatacatactaagggtttcttatatgctcaacacatgagcgaaaccctataggaaccctaattcccttatctgggaactactcacacattattatggagaaactcgagcttgtcgatcgacagatccggtcggcatctactctatttctttgccctcggacgagtgctggggcgtcggtttccactatcggcgagtacttctacacagccatcggtccagacggccgcgcttctgcgggcgatttgtgtacgcccgacagtcccggctccggatcggacgattgcgtcgcatcgaccctgcgcccaagctgcatcatcgaaattgccgtcaaccaagctctgatagagttggtcaagaccaatgcggagcatatacgcccggagtcgtggcgatcctgcaagctccggatgcctccgctcgaagtagcgcgtctgctgctccatacaagccaaccacggcctccagaagaagatgttggcgacctcgtattgggaatccccgaacatcgcctcgctccagtcaatgaccgctgttatgcggccattgtccgtcaggacattgttggagccgaaatccgcgtgcacgaggtgccggacttcggggcagtcctcggcccaaagcatcagctcatcgagagcctgcgcgacggacgcactgacggtgtcgtccatcacagtttgccagtgatacacatggggatcagcaatcgcgcatatgaaatcacgccatgtagtgtattgaccgattccttgcggtccgaatgggccgaacccgctcgtctggctaagatcggccgcagcgatcgcatccatagcctccgcgaccggttgtagaacagcgggcagttcggtttcaggcaggtcttgcaacgtgacaccctgtgcacggcgggagatgcaataggtcaggctctcgctaaactccccaatgtcaagcacttccggaatcgggagcgcggccgatgcaaagtgccgataaacataacgatctttgtagaaaccatcggcgcagctatttacccgcaggacatatccacgccctcctacatcgaagctgaaagcacgagattcttcgccctccgagagctgcatcaggtcggagacgctgtcgaacttttcgatcagaaacttctcgacagacgtcgcggtgagttcaggctttttcatatctcattgccccccgggatctgcgaaagctcgagagagatagatttgtagagagagactggtgatttcagcgtgtcctctccaaatgaaatgaacttccttatatagaggaaggtcttgcgaaggatagtgggattgtgcgtcatcccttacgtcagtggagatatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttatcacatcaatccacttgctttgaagacgtggttggaacgtcttctttttccacgatgctcctcgtgggtgggggtccatctttgggaccactgtcggcagaggcatcttgaacgatagcctttcctttatcgcaatgatggcatttgtaggtgccaccttccttttctactgtccttttgatgaagtgacagatagctgggcaatggaatccgaggaggtttcccgatattaccctttgttgaaaagtctcaatagccctttggtcttctgagactgtatctttgatattcttggagtagacgagagtgtcgtgctccaccatgttggcaagctgctctagccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattac
pCass4-Rz sequences(SEQ ID No.3):
GAATTCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAGAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGATGGCTTCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTACCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAACATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATGGTCAACATGGTGGAGCACGACACTCTCGTCTACTCCAAGAATATCAAAGATACAGTCTCAGAAGACCAGAGGGCTATTGAGACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTCATCGAAAGGACAGTAGAAAAGGAAGATGGCTTCTACAAATGCCATCATTGCGATAAAGGAAAGGCTATCGTTCAAGATGCCTCTACCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAACATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGCCTGGGTACCTCTAGAGGATCCGATACCCTGTCACCGGATGTGTTTTCCGGTCTGATGAGTCCGTGAGGACGAAACAGGACTGTCCTGCAGGAGCTCGAATTCGGTACGCTGAAATCACCAGTCTCTCTCTACAAATCTATCTCTCTCTATTTTCTCCATAAATAATGTGTGAGTAGTTTCCGATAAGGAAATTAGGGTTCTTATAGGGTTTCGTCATGTGTTGAGCATATAAGAAACCCTTAGTATGTATTTGTATTTGTAAAATACTTCTATCAATAAAATTTCTAATTCCTAAAACCAAAATCAGTACTAAATCAGATCCCCAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGTAAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGTCAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGACCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTCCCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGCTGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTCCGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCTTCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTAATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCCGATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGATAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGACGCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCAAAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCATGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTCCGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACCTTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGATCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTTCCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATAACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATACCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATAGTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACAGATCCGGTCGGCATCTACTCTATTTCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGAGTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGATTTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCATCGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCTGATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGTCGTGGCGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGCTCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGTATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTATGCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACGAGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGAGAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCAGTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATGTAGTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCTGGCTAAGATCGGCCGCAGCGATCGCATCCATAGCCTCCGCGACCGGTTGTAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCCTGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTAAACTCCCCAATGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATAAACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGGACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGCCCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAGAAACTTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATATCTCATTGCCCCCCGGGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC
Y1 fragment sequences(SEQ ID No.8)
gccgtgtgcgagacaCCGCGGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCCCGCGGccgccggcgttgtgg
Y2 fragment sequences(SEQ ID No.9)
gttgctgcctgtgatcaCCGCGGCACATTTCCCCGAAAAGTGCCACCTGAACGAAGCATCTGTGCTTCATTTTGTAGAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAAAGCGCTATTTTACCAACGAAGAATCTGTGCTTCATTTTTGTAAAACAAAAATGCAACGCGAGAGCGCTAATTTTTCAAACAAAGAATCTGAGCTGCATTTTTACAGAACAGAAATGCAACGCGAGAGCGCTATTTTACCAACAAAGAATCTATACTTCTTTTTTGTTCTACAAAAATGCATCCCGAGAGCGCTATTTTTCTAACAAAGCATCTTAGATTACTTTTTTTCTCCTTTGTGCGCTCTATAATGCAGTCTCTTGATAACTTTTTGCACTGTAGGTCCGTTAAGGTTAGAAGAAGGCTACTTTGGTGTCTATTTTCTCTTCCATAAAAAAAGCCTGACTCCACTTCCCGCGTTTACTGATTACTAGCGAAGCTGCGGGTGCATTTTTTCAAGATAAAGGCATCCCCGATTATATTCTATACCGATGTGGATTGCGCATACTTTGTGAACAGAAAGTGATAGCGTTGATGATTCTTCATTGGTCAGAAAATTATGAACGGTTTCTTCTATTTTGTCTCTATATACTACGTATAGGAAATGTTTACATTTTCGTATTGTTTTCGATTCACTCTATGAATAGTTCTTACTACAATTTTTTTGTCTAAAGAGTAATACTAGAGATAAACATAAAAAATGTAGAGGTCGAGTTTAGATGCAAGTTCAAGGAGCGAAAGGTGGATGGGTAGGTTATATAGGGATATAGCACAGAGATATATAGCAAAGAGATACTTTTGAGCAATGTTTGTGGAAGCGGTATTCGCAATATTTTAGTAGCTCGTTACAGTCCGGTGCGTTTTTGGTTTTTTGAAAGTGCGTCTTCAGAGCGCTTTTGGTTTTCAAAAGCGCTCTGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCAAAGCGTTTCCGAAAACGAGCGCTTCCGAAAATGCAACGCGAGCTGCGCACATACAGCTCACTGTTCACGTCGCACCTATATCTGCGTGTTGCCTGTATATATATATACATGAGAAGAACGGCATAGTGCGTGTTTATGCTTAAATGCGTACTTATATGCGTCTATTTATGTAGGATGAAAGGTAGTCTAGTACCTCCTGTGATATTATCCCATTCCATGCGGGGTATCGTATGCTTCCTTCAGCACTACCCTTTAGCTGTTCTATATGCTGCCACTCCTCAATTGGATTAGTCTCATCCTTCAATGCTATCATTTCCTTTGATATTGGATCATACTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAGATCAACGACATTACTATATATATAATATAGGAAGCATTTAATAGAACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCGATCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCCCGCGGtttcaaaatcggctccgtcg

Claims (10)

1. The escherichia coli-yeast-agrobacterium ternary shuttle vector is characterized in that the ternary shuttle vector is obtained by recombining yeast DNA replicons into an escherichia coli-agrobacterium binary shuttle vector, and the escherichia coli-agrobacterium binary shuttle vector is pCB301 or pCass4-Rz; the pCB301 or pCass4-Rz contains a DNA replicon of escherichia coli, a DNA replicon pVS1-origin and a T-DNA sequence of agrobacterium and a Kana resistance gene; the yeast DNA replicon is pGBK; the sequence of the ternary shuttle vector is shown as SEQ ID No.1 or SEQ ID No. 2.
2. The escherichia coli-yeast-agrobacterium ternary shuttle vector of claim 1, further comprising a yeast selection marker gene.
3. The escherichia coli-yeast-agrobacterium ternary shuttle vector according to claim 1, wherein the yeast selection marker gene is TRP1.
4. The escherichia coli-yeast-agrobacterium ternary shuttle vector according to claim 1, wherein the yeast selection marker gene indicates whether the target gene carried by the escherichia coli-yeast-agrobacterium ternary shuttle vector is expressed in the recipient cell via SD-Trp selective medium.
5. The method for preparing the escherichia coli-yeast-agrobacterium ternary shuttle vector according to claim 1, wherein a restriction enzyme Sac II single-enzyme digestion binary vector plasmid pCB301 plasmid, pCass-Rz plasmid is adopted to obtain a linearized vector.
6. The method for preparing the escherichia coli-yeast-agrobacterium ternary shuttle vector according to claim 5, wherein the enzyme digestion reaction system is as follows: plasmid pCB301 plasmid, pCass4-Rz plasma id 3. Mu.L, restriction enzyme Sac II 0.5. Mu.L, 10 XT Buffer 2. Mu.L, BSA 2. Mu.L, ddH 2 O12.5. Mu.L; cleavage reaction conditions: reacting for 12h at 37 ℃; and recovering the target linearization carrier by ethanol precipitation, and checking the correctness of the strip by electrophoresis.
7. The method for preparing the escherichia coli-yeast-agrobacterium ternary shuttle vector according to claim 1, wherein pGBKT7 plasmid is used as a template, and primers PGBK2686-F1, PGBK2686-R1, PGBK2686-F2 and PGBK2686-R2 are used for respectively performing PCR amplification to obtain 2.6Kb of two DNA fragments of Y1 and Y2 containing a yeast replicating element 2 mu sequence and a screening marker gene sequence, wherein the sequences of the primers are as follows:
forward primer PGBK2686-F1 (5 '. Fwdarw.3'):
GCCGTGTGCGAGACACCGCGGCACATTTCCCCGAAAAGTGCCACC；
reverse primer PGBK2686-R1 (5 '. Fwdarw.3'):
CCACAACGCCGGCGGCCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC；
forward primer PGBK2686-F2 (5 '. Fwdarw.3'):
GTTGCTGCCTGTGATCACCGCGGCACATTTCCCCGAAAAGTGCCACC；
reverse primer PGBK2686-R2 (5 '. Fwdarw.3'):
CGACGGAGCCGATTTTGAAACCGCGGGCGGTATTTTCTCCTTACGCATCTGTGC。
8. use of the E.coli-yeast-Agrobacterium shuttle vector according to any of claims 1 to 4 in infectious clones of plant viruses.
9. The use according to claim 8, characterized in that it comprises:
(1) Transforming a target gene and the escherichia coli-yeast-agrobacterium shuttle vector into a yeast cell, and performing yeast homologous recombination to obtain a recombinant vector;
(2) Transferring the recombinant vector containing the target gene into fungi or plant cells by an agrobacterium-mediated transformation method, and successfully detecting the expression of the target gene.
10. The use according to claim 9, wherein plasmid pCA4Y is digested with restriction enzymes Stu I, bam I to obtain pCA4Y linearized vector; the genome of the full-length cDNA of the cucumber mosaic virus is amplified in a segmented way by taking the full-length cDNA of the cucumber mosaic virus as a template, and CMV-Fny/RNA1, RNA2, yeast/StuI/F, CMV-Fny/RNA1, yeast/BamHI/R, CMV-Fny/RNA2, yeast/BamHI/R, CMV-Fny/RNA3, yeast/StuI/F, CMV-Fny/RNA3, yeast/BamHI/R as primers to obtain CMV-RNA1, RNA2 and RNA3 fragments; the base sequences of the primers of each pair are as follows:
the forward primer CMV-Fny/RNA1& RNA 2/year/StuI/F (5 '. Fwdarw.3'):
GTTCATTTCATTTGGAGAGGGTTTATTTACAAGAGCGTACGGTTCAATCC；
reverse primer CMV-Fny/RNA 1/year/BamHI/R (5 '. Fwdarw.3'):
CATCCGGTGACAGGGTATCGTGGTCTCCTTTTAGAGACCCCCACGAAAG；
reverse primer CMV-Fny/RNA 2/year/BamHI/R (5 '. Fwdarw.3'):
CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCACAAAAG；
the forward primer CMV-Fny/RNA3/yeast/StuI/F (5 '. Fwdarw.3'):
GTTCATTTCATTTGGAGAGGGTAATCTTACCACTGTGTGTGTGCGTG(；
reverse primer CMV-Fny/RNA 3/year/BamHI/R (5 '. Fwdarw.3'):
CATCCGGTGACAGGGTATCGTGGTCTCCTTTTGGAGGCCCCCACG。
CN202211258827.3A 2022-10-14 2022-10-14 E.coli-yeast-agrobacterium ternary shuttle vector and application thereof in plant virus infectious cloning Pending CN116064638A (en)

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