WO2014062036A1

WO2014062036A1 - Gene delivery system for transformation of plant using plant virus and uses thereof

Info

Publication number: WO2014062036A1
Application number: PCT/KR2013/009355
Authority: WO
Inventors: Hanhong BAE; Hyoun-Sub Lim
Original assignee: Industry Academic Cooperation Foundation of Yeungnam University
Current assignee: Industry Academic Cooperation Foundation of Yeungnam University
Priority date: 2012-10-19
Filing date: 2013-10-18
Publication date: 2014-04-24
Anticipated expiration: 2015-04-19
Also published as: KR20140050262A; KR101554678B1

Abstract

The present disclosure relates to a gene delivery system for transformation of plant using plant virus and a use thereof. It may be possible to transform a plant quickly and easily without tissue culture by using a recombinant plant expression vector of the present disclosure having a GDS cassette and a reverse transcriptase inserted into a plant virus gene. Further, the GDS cassette of the present disclosure is operable in various virus genes, and therefore, it is possible to produce transformants of various host plants, which can be infected by virus.

Description

[DESCRIPTION]

[Invention Title]

GENE DELIVERY SYSTEM FOR TRANSFORMATION OF PLANT USING PLANT VIRUS AND USES THEREOF

[Technical Field]

The present disclosure relates to a gene delivery system for transformation of a plant using a plant virus and a use thereof, and more specifically, to a cassette for plant transformation having a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence; a recombinant plant expression vector having a plant virus gene, and the cassette for plant transformation inserted into a non-coding region of the plant virus gene; a recombinant plant expression vector having a plant virus gene and a reverse transcriptase gene inserted into a random position between open reading frames (ORFs) of the plant virus gene; a method for manufacturing a transgenic plant by using the recombinant plant expression vector, wherein a target gene is inserted into the cassette for plant transformation, and the recombinant plant expression vector having a reverse transcriptase gene; a transgenic plant manufactured by the above method; and a seed thereof.

[Background Art]

Changes on climate and cultural environment caused by global warming make diseases, moisture, temperature, soil stress and the like on a plant worse. Thus, it is needed to develop various plants, which adapt to stress or have increased stress-resistance and yield. It is difficult to develop a plant adaptable to environmental change by the pre-existing breeding methods, and also in the preexisting transformation methods, processes for developing new plants adaptable to the rapidly changing environment consume time, cost and labor in large.

In general, transformation is to make express new character since continuous replication is enabled and a foreign gene is expressed by forming an independent replicable unit (replicon) by introducing the foreign gene into another cell, or integrating the foreign gene by homologous recombination into the genome of the introduced cell. For plant transformation, methods using a gene gun (particle bombardment) and an agrobacterium have been mainly used in the past, but due to high-cost and low-efficiency of the gene gun-mediated transformation method, the method using an agrobacterium is used more. The transformation method using an agrobacterium can complete a transformant by tissue culture, but this is a method whose development takes too long, and may be succeed by only skilled researchers. Further, the above methods need a tissue culture facility for completing transformants and a greenhouse facility for cultivating regeneration plants, and it is difficult to be used by common researchers in terms of the cost. Further, there are defects that it is needed to be cautious regarding fungal contamination in young plants during tissue culture, and many labors are needed to be added.

The plant virus-based vector is a useful tool for effectively expressing a target protein in a plant. The plant virus-based expression system is more advantageous than other methods for producing a recombinant protein The reason is that it is a method, which is cheaper and easier to cultivate than tissue culture, thereby directly applicable to a plant tissue culture, and wherein a foreign gene is rapidly expressed. However, this system is suitable for temporarily expressing a foreign gene in a plant.

The present inventors provide an easy and quick gene delivery system using a plant virus, as a method for replacing the pre-existing transformation methods, and therefore, provide a technique enabled for manufacturing various crops, to which crops and useful characters adaptable to climatic and environmental change are introduced.

On the other hand, Korean Patent Registration No.1122955 discloses 'methods for overexpression of foreign genes in a plant using infectious clones of soybean mosaic virus', and Korean Patent Publication No.2006-0013000 discloses 'a plant expression vector containing BCTV replicon'. However, there is no mention about a gene delivery system for transformation of plant using a plant virus, and a use thereof, as described in the present disclosure.

[Disclosure]

[Technical Problem]

The present disclosure is designed to solve the above demands. Therefore, the present disclosure was completed by developing a gene delivery system (GDS) cassette, which includes a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence, and can be inserted into a plant virus gene; and confirming that a target gene inserted into a GDS cassette is integrated into a plant chromosome and expressed by using a recombinant plant expression vector including the GDS cassette and a reverse transcriptase inserted into the plant virus gene. [Technical Solution]

In order to solve the problems, the present disclosure provides a cassette for plant transformation including a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence, in the 5' to 3' direction.

Further, the present disclosure provides a recombinant plant expression vector including a plant virus gene and the cassette for plant transformation inserted into a non-coding region of the plant virus gene.

Further, the present disclosure provides a recombinant plant expression vector including a plant virus gene and. a reverse transcriptase gene inserted into a random position between open reading frames (ORFs) of the plant virus gene.

Further, the present disclosure provides a method for manufacturing a transgenic plant expressing a target gene, which includes:

a step of inserting a target gene into the recombinant plant expression vector having the cassette for plant transformation;

a step of manufacturing a RNA transcript by cutting the recombinant plant expression vector inserted with the target gene followed by conducting in vitro transcription;

a step of inoculating the RNA transcript into a plant; and

a step of inoculating an agrobacterium, which has the recombinant plant expression vector inserted with the reverse transcriptase gene, into the plant.

a step of inserting a target gene into the recombinant plant expression vector having the cassette for plant transformation and a reverse transcriptase gene;

a step of transforming an agrobacterium with the recombinant plant expression vector inserted with the target gene; and

a step of inoculating the transformed agrobacterium into a plant.

Further, the present disclosure provides a transgenic plant manufactured by the above method, and a seed thereof.

[Advantageous Effects]

According to the method for transforming a plant by using a recombinant plant expression vector having a GDS cassette and a reverse transcriptase inserted into a plant virus gene, it is possible to transform a plant quickly and easily without tissue culture. Further, the GDS cassette of the present disclosure is operable in various virus genes, and therefore, it is expected to produce various transgenic plants by using viral host plants.

[Description of Drawings]

Fig. 1 shows a cassette, in which a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence are inserted from 5' to 3' direction.

Fig. 2 shows a vector having CaMV reverse transcriptase and [polypurine tract-LB-CaMV35S-MCS-NOSterRB-t ^et] cassette.

Fig. 3 shows a polypurine tract as a transcription initiation sequence of the synthesized CaMV reverse transcriptase and a tRNA Met binding sequence.

Fig. 4 shows [LB-CaMV35S-MCS-NOS_ter-RB] cassette inserted between a polypurine tract and a tRNA Met binding sequence.

Fig. 5 shows the result confirming whether eGFP cDNA is produced or not by inoculating an agrobacterium, which is transformed with PVX, i.e., eGFP-inserted RNA virus, and pCambia:CaMV RT, into a tobacco plant.

Fig. 6 shows the result confirming whether CaMV reverse transcriptase and a cassette gene is inserted or not by inoculating a vector, in which the CaMV reverse transcriptase is inserted between P1 and HC-Pro of SMV, and a SMV infectious clone inserted with [polypurine tract-LB-CaMV35S-MCS-NOSter-RB-tMet] cassette, into a plant.

Fig. 7 shows the result confirming that eGFP is expressed in Arabidopsis thalian by [polypurine tract-LB-CaMV35S-eGFP-NOS_terRB-t ^et] cassette and CaMV reverse transcriptase inserted into alternanthera mosaic virus (AltMV) gene.

[Best Mode]

In order to accomplish the objects of the present disclosure, the present disclosure provides a cassette for plant transformation including a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence from 5' to 3' direction.

The cassette for plant transformation of the present disclosure refers to a DNA set to enable the expression of a protein desired to be expressed by transforming a plant, and the cassette used in the present disclosure includes a DNA sequence consisting of a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS) for target gene insertion, a terminator, a right border (RB) and a tRNA Met binding sequence. The MCS is operably linked to the promoter. In the present disclosure, the term "operably linked" refers to a functional connection between a promoter sequence and a target gene, which is inserted in a MCS controlled by the promoter sequence. The operably linked promoter controls the expression of a polypeptide encoded by a target gene.

In the cassette for plant transformation of the present disclosure, the polypurine tract is a ubiquitous nucleotide sequence appeared at some region of a genomic DNA, wherein purine bases are aligned at one DNA strand, and pyrimidine bases are aligned at the other DNA strand. In the present disclosure, the polypurine tract reacts with a reverse transcriptase, thereby working as a transcription initiation sequence. The polypurine tract may consist of the nucleotide sequence of SEQ ID NO. 1 , but not limited thereto. Further, homologues of the nucleotide sequence may be included within the scope of the present disclosure. The homologues are nucleotide sequences, whose nucleotide sequence may be changed, but which have similar functional characteristics with the nucleotide sequence of SEQ ID NO. 1. Specifically, the genes may include a nucleotide sequence having sequence homology of at least 70%, more specifically, at least 80%, still more specifically, at least 90%, and most specifically, at least 95% with the nucleotide sequence of SEQ ID NO. 1 , respectively. The "% of sequence homology" for a polynucleotide may be confirmed by comparing two nucleotide sequences that are optimally arranged with a region to be compared. In this regard, a part of the polynucleotide sequence in a region to be compared may include an addition or a deletion (i.e., gap) compared to a reference sequence (without any addition or deletion) relative to the optimized arrangement of the two sequences.

The Left border (LB) and the Right border (RB) used in the cassette for plant transformation of the present disclosure are located at the front/rear of MCS, where a target gene may be inserted, and make the target gene to be integrated to a plant genome. The LB may consist of the nucleotide sequence of SEQ ID NO. 4, but not limited thereto, and homologues of the nucleotide sequence may be included within the scope of the present disclosure. Further, the RB may consist of the nucleotide sequence of SEQ ID NO. 5, but not limited thereto, and homologues of the nucleotide sequence may be included within the scope of the present disclosure. The MCS indicates a DNA fragment having restriction sites, which are recognized by various restriction enzymes and cut, and therefore, it may be possible to insert a target gene into the MCS site, which is recognized and cut by a certain restriction enzyme. The MCS may be any MCS known to those skilled in the art of the present disclosure, without limitation, and for example, it may be a DNA fragment including restriction enzyme sites of Mlu\, BamH\ and Nhe\, but not limited thereto. It is also possible to add restriction enzyme sites, which may be commonly used in the art, other than the tree types of restriction enzyme sites described above to the MCS. When inserting a target gene into the MCS of the cassette of the present disclosure, a restriction enzyme site, which does not exist in a plant virus gene, may be used. The sequences of the plant virus genes are different each other, and therefore, restriction enzyme sites in the MCS to be used may vary depending on the type of the plant virus.

In the cassette for plant transformation of the present disclosure, the promoter may be a promoter suitable for transformation, and for example, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, histone promoter or Clp promoter, but not limited thereto. The term "promoter" refers to a region of DNA located upstream of a structure gene, and it corresponds to a DNA molecule to which an RNA polymerase binds to initiate transcription. The term "plant promoter" refers to a promoter that may initiate transcription in a plant cell. The term "constitutive promoter" refers to a promoter that is active under most environmental conditions and cell growth or differentiation state. Since selection of a transformant may be made for various tissues at various stages, the constitutive promoter may be preferred for the present disclosure. Thus, selection possibility is not limited by the constitutive promoter.

In the cassette for plant transformation of the present disclosure, the terminator may be any typical terminator, for example, nopalin synthase (NOS), rice a-amylase RAmyl A terminator, a terminator for Octopine gene of Agrobacterium tumefaciens, phaseoline terminator, rrnB1/B2 terminator of E. Coli and the like, but not limited thereto. Regarding the necessity of terminator, it is generally known that the terminator region may increase reliability and efficiency of gene transcription in plant cells. Therefore, the use of a terminator is highly preferable in view of the context of the present disclosure.

In the cassette for plant transformation of the present disclosure, the tRNA Met binding sequence plays a role of a primer for a reverse transcriptase, thereby enabling cDNA synthesis by the reverse transcriptase. The tRNA Met binding sequence may consist of the nucleotide sequence of SEQ ID NO. 2, but it may be the sequence, which may work as a primer for a reverse transcriptase, without limitation. Further, homologues of the nucleotide sequence may be included within the scope of the present disclosure.

In one embodiment of the present disclosure, the cassette for plant transformation may be the cassette shown in FIG. 1 , but not limited thereto.

In one embodiment of the present disclosure, the cassette for plant transformation may consist of the nucleotide sequence of SEQ ID NO. 3, but not limited thereto. Further, homologues of the nucleotide sequence may be included within the scope of the present disclosure.

In one embodiment of the present disclosure, the recombinant plant expression vector of the present disclosure including the cassette for plant transformation may further include a reverse transcriptase gene, which is inserted into a random position between open reading frames (ORFs) of the plant virus gene.

Further, the present disclosure provides a recombinant plant expression vector including a plant virus gene and the reverse transcriptase gene, inserted into a random position between open reading frames (ORFs) of the plant virus gene.

In the present disclosure, the non-coding region of the plant virus gene, where the cassette for plant transformation is inserted, is a region excluding a transcription regulation region, and specifically, it may be a 3' non-coding region, but not limited thereto. Further, the random position between ORFs of the plant virus, where the reverse transcriptase gene is inserted, does not affect to the expression of viral proteins, and it is a region, where the reverse transcriptase may be expressed. Specifically, it may be located between a protease 1 (P1) coding gene and a virus infection specific donor (helper component/protease; HC-Pro) coding gene of a plant virus, but not limited thereto. The specific recombinant plant expression vector of the present disclosure may include a vector having a plant virus gene inserted with the cassette for plant transformation and a vector having the same plant virus gene inserted with a reverse transcriptase separately, or it may include a plant virus gene, the cassette for plant transformation inserted into a non-coding region of a plant virus gene and a reverse transcriptase gene inserted into a random position between ORFs of the plant virus gene in one vector. The plant virus may be any plant virus, which may be infected into a host plant and proliferate therein, without limitation. Namely, the kind of the plant virus may differ depending on each host plant. Specifically, the plant virus may be soybean mosaic virus (SMV), or potato virus X (PVX) or alternanthera mosaic virus (AltMV) belongs to potexvirus, but not limited thereto.

The term "recombinant" indicates a cell which replicates a heterogeneous nucleotide or expresses the nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. A recombinant cell may express a gene or a gene fragment in a form of a sense or antisense, which is not found in natural state of cell. In addition, a recombinant cell can express a gene that is found in the natural state, but the gene may be modified and re-introduced into the cell by an artificial means.

In the present disclosure, the cassette for plant transformation or a reverse transcriptase gene sequence may be inserted into a recombinant vector. The term "vector" is used herein to refer DNA fragment(s) and nucleotide molecules that are delivered to a cell. The vector may replicate DNA, and be independently reproduced in a host cell. The term "delivery system" and the term "vector" are often interchangeably used. The recombinant vector indicates bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell viral vector, or other vectors. In general, random plasmid and vector may be used if they may be replicated and stabilized in a host. An important characteristic of the expression vector is having an origin of replication, a promoter, a marker gene and a translation control element.

The expression vector, which includes each sequence and proper transcription/translation regulation signal of the cassette for plant transformation or the reverse transcriptase gene, may be constructed by a method known to those skilled in the art. The method may include in vitro recombinant DNA technique, DNA synthesis technique, in vivo recombination technique and the like. The DNA sequence may be effectively linked to a proper promoter in the expression vector, in order to lead mRNA synthesis. Further, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

A specific example of the recombinant vector of the present disclosure may be Ti-plasmid vector which may transfer a part of itself, i.e., so-called T-region, to a plant cell when the vector is present in an appropriate host such as Agrobacterium tumefaciens. Other types of the Ti-plasmid vector (see, EP 0 116 718 B1 ) are currently used for transferring a hybrid DNA sequence to a plant cell or protoplasts that may produce a new plant by appropriately inserting hybrid DNA into a plant genome. Another specific form of the Ti-plasmid vector is a so-called binary vector which has been disclosed in EP 0 120 516 B 1 and U.S. Pat. No. 4,940,838. The binary vector, which may be used in the present disclosure, may be any binary vector including a RB and LB of a T-DNA, which may transform a plant when existing with the Ti plasmid of agrobacterium tumefaciens, and specifically, it may be pBI101 (Cat#: 6018-1 , clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121 , pCAMBIA vector and the like, which is often used in the art. Other appropriate vectors that may be used for introducing the DNA into a plant host may be selected from a double-stranded plant virus (for example, CaMV), a single-stranded virus, and a viral vectors, which may be derived from Gemini virus and the like, for example, a non-complete plant viral vector. Use of the vector may be especially advantageous when it is difficult to properly transform a plant host.

In the recombinant plant expression vector of the present disclosure, the promoter may be a promoter suitable for in vitro transcription or transformation, and specifically, it may be T7 promoter, SP6 promoter, CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter, histone promoter or Clp promoter, more specifically, T7 promoter, SP6 promoter or CaMV 35S promoter, but not limited thereto.

In the recombinant plant expression vector of the present disclosure, the terminator may be any typical terminator increasing a reliability and an efficiency of gene transcription in plant cells, for example, nopalin synthase (NOS), rice a-amylase RAmyl A terminator, a terminator for Octopine gene of Agrobacterium tumefaciens, phaseoline terminator, rrnB1/B2 terminator of E.Coli and the like, but not limited thereto.

Specifically, the recombinant vector may include at least one selection marker. The marker is a nucleotide sequence having a property which allows a selection based on a common chemical method, and it may be any kind of gene that may be used for distinguishing transformed cells from non-transformed cells. For example, it may be a gene resistant to herbicide such as glyphosate and phosphinotricin, and a gene resistant to antibiotics such as kanamycin, hygromycin, chloramphenicol, G418 and bleomycin, but not limited thereto.

a step of manufacturing a RNA transcript by cutting the target gene-inserted recombinant plant expression vector followed by conducting in vitro transcription; a step of inoculating the RNA transcript into a plant; and

a step of inoculating an agrobacterium, which includes the reverse transcriptase gene-inserted recombinant plant expression vector, into the plant.

In the method for manufacturing a transgenic plant of the present disclosure, the in vitro transcription for manufacturing the RNA transcript may use, for example, T7 RNA polymerase, but not limited thereto.

a step of transforming an agrobacterium with the target gene-inserted recombinant plant expression vector; and

a step of inoculating the transformed agrobacterium into a plant.

There is no limit to the target gene, which may be introduced into the vector of the present disclosure. In Examples of the present disclosure, a reporter gene, enhanced green fluorescent protein (eGFP) gene is used instead of the target gene. The GFP may be observed through green fluorescence by long wavelength UV irradiation, and therefore, it may be very useful for confirming the transformed plant.

The method for manufacturing a transgenic plant of the present disclosure may be a method for transforming a vector including a plant virus gene inserted with the cassette for plant transformation and a vector including the same plant virus gene inserted with a reverse transcriptase, respectively, or a method for transforming one vector including a plant virus gene, the cassette for plant transformation inserted into a non-coding region of the plant virus gene and a reverse transcriptase gene inserted into a random position between ORFs of the plant virus gene.

Specifically, in the method for transforming a plant by using the recombinant plant expression vector of the present disclosure, a target gene to be expressed is inserted into a MCS of the recombinant plant expression vector, the target gene- inserted recombinant plant expression vector is cut, in vitro transcription is conducted to prepare a RNA transcript, and then the RNA transcript is inoculated into a plant. Then, the recombinant plant expression vector including a plant virus gene, and a reverse transcriptase gene inserted into a random position between open reading frames (ORFs) of the plant virus gene is transformed into an agrobacterium, the transformed agrobacterium is inoculated into the RNA transcript-inoculated plant, and thereby, the produced reverse transcriptase synthesizes cDNA by using a tRNA Met binding sequence existing in the RNA transcript as a primer. The synthesized cDNA includes a LB and a RB. Accordingly, the LB and the RB insert a sequence containing the target gene between the LB and the RB into a chromosome of a plant, and thereby, the target gene may be transformed into the plant.

In these processes, the reverse transcriptase gene may be included in a separate recombinant plant expression vector, and may be also inserted into the recombinant plant expression vector inserted with the cassette for plant transformation of the present disclosure. In the later case, specifically, the method transforming a plant by using the recombinant plant expression vector of the present disclosure includes a step of inserting a target gene to be expressed into a MCS of the recombinant plant expression vector, a step of transforming an agrobacterium with the target gene-inserted recombinant plant expression vector, and a step of inoculating the transformed agrobacterium into a plant.

The plant virus may be a plant virus, which may infect a host plant and proliferate therein, and specifically, it may be any plant virus, which may infect a plant to be transformed and proliferate therein, without limitation. Specifically, it may be soybean mosaic virus (SMV), or potato virus X (PVX) or alternanthera mosaic virus (AltMV) belongs to potexvirus, but not limited thereto.

The method for manufacturing a transgenic plant of the present disclosure may be conducted by inoculating an agrobacterium including the RNA transcript of the target gene-inserted cassette for plant transformation and the reverse transcriptase gene-inserted recombinant plant expression vector, into a mature plant, or by inoculating an agrobacterium including the recombinant plant expression vector, to which the reverse transcriptase gene- and the target gene-inserted cassette for plant transformation is inserted, into a mature plant. Thus, this method does not need tissue culture of a plant cell, and therefore, it does not need a step of regenerating a transformed plant from the plant cell.

Further, the present disclosure provides a transgenic plant manufactured by the above method and a seed thereof.

In one embodiment of the present disclosure, the plant may be a dicotyledon or a monocotyledon, and it may be any host plant, to which a plant virus may infect, and in which the virus may proliferate, without limitation. Specifically, the plant may be: food crops selected from the group consisting of rice, wheat, barley, corn, soybean, potato, red bean, oats and millet; vegetable crops selected from the group consisting of Arabidopsis thalian, Chinese cabbage, radish, pepper, strawberry, tomato, water melon, cucumber, cabbage, oriental melon, pumpkin, Welsh onion, onion and carrot; specialty crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugarcane, sugar beet, perilla, peanut and canola; fruit trees selected from the group consisting of apple tree, pear tree, jujube tree, peach, kiwi, grape, tangerine, persimmon, plum, apricot and banana; flowering plants selected from the group consisting of rose, gladiola, gerbera, carnation, chrysanthemum, lily and tulip; and feed crops selected from the group consisting of rye grass, red clover, orchard grass, alfalfa, tall fescue, and perennial rye grass, and more specifically, it may be bean, potato or Arabidopsis thalian, but not limited thereto.

The examples and experiments will now be described. The following examples and experiments are for illustrative purposes only and not intended to limit the scope of the present disclosure.

Example 1. Evaluation of Ca V Transcription Initiation Sequence

By using a plant virus, which is free to move in a plant, an experiment for developing a gene delivery system (GDS), which makes crop transformation easy, is conducted. GDS vectors are manufactured by using reverse transcriptases of soybean mosaic virus (SMV) for bean transformation, and potexvirus (PVX), alternanthera mosaic virus (AltMV) and cauliflower mosaic virus (CaMV) for vegetable transformation.

Prior to manufacture of a GDS vector, in order to obtain a polypurine tract as a CaMV reverse transcriptase transcription initiation sequence and a tRNA Met binding sequence, a double-strand is formed by treating synthetic oligomers, Poly-P- Top (SEQ ID NO. 6) and Poly-P-Bottom (SEQ ID NO. 7), at 95°C for 5 min followed by maintaining at room temperature for 10 min, and after adding Nco\ sites (nullified) to the 5' and 3' ends, inserted into the Nco\ site of pUC-ALMCS vector. The inserted nucleotide sequence is confirmed, and it is named pUC-AI-RT-ln (FIG. 3).

CaMV reverse transcriptase is amplified with pGD-RT-Mlul-F (SEQ ID NO. 8) and pGD-RT-Xmal-R (SEQ ID NO. 9) primers, and then cloned into a pCAMBIA binary vector, thereby pCambia:CaMV RT is produced. In order to evaluate the synthesized polypurine tract, eGFP is amplified with eGFP-BamHI-F (SEQ ID NO. 10) and eGFP-Mlul-R (SEQ ID NO. 11), inserted between the polypurine tract and the tRNA Met binding sequence of pUC-AI-RT-lnrine tract, and then cloned into a potexvirus, PVX (FIG. 5).

The PVX, the eGFP-inserted RNA virus, is inoculated into a tobacco plant in advance, and two weeks later, an agrobacterium transformed with pCambia:CaMV RT is inoculated thereinto by agroinfiltration method. In order to confirm whether eGFP cDNA is produced or not, DNA is extracted from a plant, all RNA is removed with RNase, and then PCR is conducted by using the eGFP primers. The eGFP is amplified at the DNA extracted from the plant, treated with the eGFP-inserted PVX and the pCambia:CaMV RT, but it is not amplified at a control group not treated with the pCambia:CaMV RT (FIG. 5). In a RNA virus, the eGFP is transcribed as only RNA, but the eGFP, inserted between the polypurine tract as a CaMV reverse transcriptase transcription initiation sequence and the tRNA Met binding sequence, is converted to cDNA by means of the CaMV reverse transcriptase. It is confirmed that the polypurine tract synthesized through the above process is a structure enabling reverse transcription.

Example 2. Manufacture of Cassette for Plant Transformation and Vector After confirming that the CaMV reverse transcriptase is reacted with the polypurine tract, a vector including the cassette, in which a left border (LB), CaMV35S promoter, a multiple cloning site (MCS), NOS terminator and a right border (RB) [LB-CaMV35S-MCS-NOSter-RB] sequences are inserted between the polypurine tract and the tRNA Met binding sequence, is manufactured. A GDS vector designed for inserting a target gene, which is converted to cDNA by using a plant virus delivering a target gene to a nucleus of a plant cell and CaMV reverse transcriptase, into a chromosome of a plant cell, is produced by inserting the LB (TGGTGGCAGGATATATTGTGGTGTAAACAA: SEQ ID NO. 4) and the RB (GTAAACCTAAGAGAAAAGAGCGTTAT: SEQ ID NO. 5) sequences at the front/rear of the MCS for cloning the target gene, respectively. Further, for the stable expression of the target gene, the CaMV35S promoter and the NOS terminator sequences are added (FIG. 1).

In order to add LB-CaMV35S-MCS-NOSter-RB to the polypurine tract nucleotide sequence, a region from the LB to the RB of the binary vector is amplified by using Mlu-F-LB (SEQ ID NO. 12) and Nhel-R-RB (SEQ ID NO. 13) primers, and it is cloned to pUC-AI-RT-ln (FIG. 3) to produce pUC-LB-MCS-RB (FIG. 4). As MCS, Xho\, BglU, HindW and BamH\ existing in the binary vector are maintained, so as to make the target gene to be cloned easily.

The produced GDS vector includes the cassette, to which the polypurine tract as a CaMV reverse transcriptase transcription initiation sequence, LB, CaMV35S promoter, MCS, NOS terminator, RB and tRNA Met binding sequence working as a primer of the CaMV reverse transcriptase [polypurine tract-LB-CaMV35S-MCS- NOS_ter-RB-t^Met] are inserted (FIG. 1 ). The cassette sequence is in the size of about 1.3 kb, and is manufactured for being inserted to potexvirus or alternanthera mosaic virus (AltMV) and the like as well as soybean mosaic virus (SMV), so as to be applied to various crops.

Example 3. Manufacture of Soybean Mosaic Virus GDS vector

In order to express the CaMV reverse transcriptase and the [polypurine tract- LB-CaMV35S-MCS-NOS_ter-RB-t^Met] cassette in a soybean mosaic virus (SMV) gene, a SMV GDS vector is manufactured by using a SMV infectious clone.

In order to insert the CaMV reverse transcriptase between a SMV protease 1 (P1) coding gene and a virus infection specific donor (helper component/protease; HC-Pro) coding gene, the CaMV reverse transcriptase is amplified with SMV-Mlul-F- RT (SEQ ID NO. 14) and SMV-Mlul-R-RT (SEQ ID NO. 15) primers. The CaMV reverse transcriptase inserted between the P1 and the HC-Pro is cloned into a binary vector, pGD vector (SMV-CaMV-RT, FIG. 2). Because the RNA virus, SMV produces polyprotein, and the polyprotein is converted to a functional protein after the virus protease cut the polyprotein, a SMV protease recognition amino acid, Threonine-Arginine sequence is involved to the CaMV reverse transcriptase inserted between the P1 and the HC-Pro.

The [polypurine tract-LB-CaMV35S-MCS-NOS_terRB-t^Met] cassette is amplified with SMV-LB-Xbal-F (SEQ ID NO. 16) and SMV-RB-Xbal-R (SEQ ID NO. 17) primers, inserted at the Xba\ site of the 3' non-coding region of the SMV, and then cloned into a pTOP V2 vector. In order to inoculate the SMV infectious clone inserted with the [polypurine tract-LB-CaMV35S-MCS-NOS_ter-RB-t^Met] cassette with the RNA transcript obtained by in vitro transcription excluding DNA inoculation, the CaMV35S promoter of the pTOP V2 vector is removed, and replaced with a T7 promoter (SMV-LB-MCS-RB, FIG. 2).

The manufactured SMV-CaMV-RT and SMV-LB-MCS-RB are inoculated to Jack cultivar in the forms of plasmid DNA and RNA transcript, respectively, and then the viral symptom and the expression of the inserted gene are confirmed (FIG. 6). This proves that a reverse transcriptase is expressed in a virus, and it also proves that the LB-CaMV35S-MCS-NOSter-RB is operable in a plant virus.

Example 4. Manufacture of GDS Vector for Arabidopsis thalian Using AltMV In order to develop a gene delivery system for a model plant, Arabidopsis thalian, a GDS vector, wherein the [polypurine tract-LB-CaMV35S-MCS-NOSter-RB- tMet] cassette is inserted into alternanthera mosaic virus (AltMV) gene (GenBank No.

GQ 179646) using various vegetables as a host plant, is manufactured.

The [polypurine tract-LB-CaMV35S-MCS-NOSter-RB-tMet] cassette sequence is amplified with AltMV-LB-Ncol (SEQ ID NO. 18) and AltMV-RB-Ncol

(SEQ ID NO. 19) primers, and then cloned to the Nco\ site of the pUC-AltMV-MCS to manufacture pUC-AltMV-LB-MCS-RB. The manufactured pUC-AltMV-LB-MCS-RB is inoculated to Arabidopsis thalian, and then infectiousness is confirmed by PCR. eGFP is amplified with LB-eGFP-Xhol-F (SEQ ID NO. 20) and RB-eGFP-BamHI-R (SEQ ID NO. 21) primers, and then cloned to the Xho\ and BamHl sites of the MCS to manufacture pUC-LB-eGFP-RB finally. LB-eGFP-RB is amplified with AltMV-LB- Mlul-F (SEQ ID NO. 22) and AltMV-RB-Ncol (SEQ ID NO. 23) primers, and then cloned to the Nco\ and Mlu\ sites of pGD-AltMV-MCS. In order to confirm the eGFP cDNA synthesis by using a reverse transcriptase, the [polypurine tract-LB-CaMV35S- MCS-NOSter-RB-tMet] cassette is inserted in the reverse direction (FIG. 7). The AltMV-inverted LB-eGFP-RB is linearized by Xba\, and then transcribed in vitro. The obtained RNA transcript is inoculated into Arabidopsis thalian. Two weeks later, the CaMV RT is expressed in a leaf, where symptoms are appeared, by agroinfiltration method. The eGFP expression in the Arabidopsis thalian leaf is confirmed by using a fluorescence microscope (FIG. 7). This result proves that the [polypurine tract-LB-CaMV35S-MCS-NOSter-RB-tMet] cassette is inserted to a chromosome of a cell of the Arabidopsis thalian leaf.

Claims

[CLAIMS]

[Claim 1 ]

A cassette for plant transformation comprising a polypurine tract, a left border (LB), a promoter, a multiple cloning site (MCS), a terminator, a right border (RB) and a tRNA Met binding sequence, in the 5' to 3' direction.

[Claim 2]

The cassette for plant transformation according to claim 1 , wherein the polypurine tract is consisting of the nucleotide sequence of SEQ ID NO. 1 , and the tRNA Met binding sequence is consisting of the nucleotide sequence of SEQ ID NO. 2.

[Claim 3]

The cassette for plant transformation according to claim 1 , which is a cassette illustrated in Fig. 1.

[Claim 4]

The cassette for plant transformation according to claim 1 , which is consisting of the nucleotide sequence of SEQ ID NO. 3.

[Claim 5]

A recombinant plant expression vector comprising a plant virus gene and the cassette for plant transformation of any one of claims 1 to 4 inserted into a non- coding region of the plant virus gene.

[Claim 6]

The recombinant plant expression vector according to claim 5, which further comprises a reverse transcriptase gene inserted into a random position between open reading frames (ORFs) of the plant virus gene.

[Claim 7]

A recombinant plant expression vector comprising a plant virus gene and a reverse transcriptase gene inserted into a random position between open reading frames (ORFs) of the plant virus gene.

[Claim 8]

A method for manufacturing a transgenic plant expressing a target gene, which comprises:

a step of inserting a target gene into the recombinant plant expression vector of claim 5;

a step of inoculating the RNA transcript into a plant; and

a step of inoculating an agrobacterium, which comprises the recombinant plant expression vector of claim 7, into the plant.

[Claim 9]

a step of inserting a target gene into the recombinant plant expression vector of claim 6;

a step of inoculating the transformed agrobacterium into a plant.

[Claim 10]

A transgenic plant manufactured by the method of claim 8 or 9.

[Claim 11 ]

A seed of the transgenic plant according to claim 10.