WO2009066919A2

WO2009066919A2 - Gpt gene as a marker for selecting plant transformant

Info

Publication number: WO2009066919A2
Application number: PCT/KR2008/006784
Authority: WO
Inventors: Jae Heung Jeon; Hyun Soon Kim; Yoon Sik Kim; Yong Hwa Lee; Mi Sun Kim; Sung Ran Min; Won Joong Jeong; Suk Yoon Kwon; Hyouk Joung
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-11-23
Filing date: 2008-11-18
Publication date: 2009-05-28
Also published as: WO2009066919A3; KR20090053230A; KR100927803B1

Abstract

The present invention relates to a marker consisting of GPT (UDP-Λ/-acetylgluco samine:dolichol phosphate-Λ/-acetylglucosamine-phosphotransferase) gene to select a transformed plant, a recombinant plant expression vector comprising GPT gene, plants that are transformed with said vector and seeds of the plants, a method of selecting a tr ansformed plant comprising steps of transforming a plant cell with said vector and selec ting a transformant in a medium comprising tunicamycin, and a method of producing a t ransformed plant comprising a step of transforming a plant cell with said vector.

Description

GPT GENE AS A MARKER FOR SELECTING PLANT TRANSFORMANT

TECHNICAL FIELD

The present invention relates to a method of selecting genetically modified plant s by using GPT (UDP-ZV-acetylglucosamineidolichol phosphate-Λ/-acetylglucosamine- phosphotransferase) gene originating from a plant. More specifically, the present inve ntion relates to a marker consisting of GPT gene for selecting a plant transformant, a re combinant plant expression vector comprising GPT gene, plants that are transformed w ith said vector and seeds of the plants, a method of selecting a transformed plant comp rising steps of transforming a plant cell with said vector and selecting a transformant in a medium comprising tunicamycin, and a method of producing a transformed plant com prising a step of transforming a plant cell with said vector.

BACKGROUND ART

Genetic engineering is a technology by which a gene can be isolated from one sped es and then inserted to other species. Determination of the structure of DNA by Dr. Watso n and Dr. Crick in 1953 and development of a way of digesting DNA in 1970s made possibl e to achieve a genetic engineering technology.

Since a gene which expresses a desired property can be artificially isolated and then inserted into other organism according to a genetic engineering method, it is highly likely th at such property is successfully expressed in short time. As a result, with such advantages

, a genetic engineering technology is now a very common method in the field of molecular b iology. Meanwhile, an organism which is newly produced by this genetic engineering meth od is referred to as a "genetically modified organisms (abbreviated as "GMO"). When food crops such as rice, potato, corn, bean and the like are produced by this genetic engineerin g method, they are referred to as genetically modified crops. Food products that are obtai ned by processing such genetically modified crops are referred to as genetically modified fo od products.

For genetically modified crops, a breed having good resistance to harmful insects an d weeds can be obtained in a great amount in short time with a genetic engineering technol ogy. Thus, they can be very useful not only for solving the global food shortage problem b ut also for achieving dramatic improvement of taste and nutrition of the crops or for solving a problem of a lack of proper nutrition based on incorporation of a pharmaceutical compone nt. In Korea, it is believed that about 30% of soybean which had been imported for produc ing soybean curd, soybean sauce, soybean milk and fermented soybean cake, etc. are gen etically modified soybeans. Thus, most Koreans had already experienced genetically modi fied crops and the use of the genetically modified crops will further increase in future.

Meanwhile, for specific selection of a transformed plant, a bacteria-originated marker gene such as a gene having resistance to antibiotics or herbicides and the like is used. H owever, there is a rising concern that, when outside natural environment is contaminated wi th such gene, a super weed which has high resistance to herbicides or super bacteria that c annot be terminated with regular antibiotics such as penicillin can be generated and the eco logical system can be disrupted by them. Such potential problems pose the question of w hether or not genetically modified crops can be commercially successful.

In order to obtain genetically modified crops which are free of the above-described pr oblems and have a high commercial value, an environmentally friendly gene that can be us ed as a substitute for a gene which has resistance to antibiotics or herbicides and may caus e a disturbance in an ecological system should be developed. Such efforts are absolutely required to guarantee the safety of the genetically modified food products.

GPT is a key enzyme for catalyzing an initial reaction required for the synthesis o f dolichol-p-p-oligosaccharides during dolichol pathway and the glycosylation of N-linke d glycoproteins. In addition, GPT promotes a biological synthesis of Asn-linked glycan s (Tine Kring Sorensen et al., 2003, Biochimica et Biophysica Acta, 1619: 89-97). One of the important aspects of Asn-linked glycan is that it requires precise folding of its pol ypeptide chain by chaperone molecules in endoplasmic reticulum. Misfolded proteins can induce a stress reaction such as unfolded protein response (UPR) and get involved with up-regulation of the chaperones present in endoplasmic reticulum, for example, Bi P (binding protein). UPR is induced when proteins are synthesized in the presence of amino acid analogs or tunicamycin which is an inhibitor for the formation of Asn-linked g lycans. Specifically, when Asn-linked glycosylation is inhibited by tunicamycin, transcri ption of BiP is induced and such induction eventually causes the expression of UPR. Tunicamycin is a strong inhibitor of GPT, which is the enzyme working first in the biologi cal pathway for synthesizing glycans. It inhibits the synthesis of the glycans by inhibiti ng further addition of initial polypeptides. However, it is also reported that tunicamycin does not inhibit other N-acetylglucosamine transferase (Nozomu Koizumi et al., 1999, P lant Physiology, 121: 353-361). DETAILED DESCRIPTION OF THE INVENTION Technical Problem

The present invention, which is devised in view of the above aspects, is to impro ve the safety of environmentally friendly and genetically modified plants by providing a method in which GPT gene originating from a plant is used as a selection marker and tu nicamycin is used for the selection of genetically modified plants.

Technical Solution

In order to solve the problems described in the above, the present invention provide s a marker consisting of GPT (UDP-Λ/-acetylglucosamine:dolichol phosphate-Λ/- acetylgluco samine-phosphotransferase) gene to see whether or not a plant is transformed.

Further, the present invention provides a recombinant plant expression vector compri sing GPT gene.

Further, the present invention provides plants that are transformed with said vector a nd seeds of the plants.

Further, the present invention provides a method of selecting a transformed plant co mprising steps of transforming a plant cell with said vector and selecting a transformant in a medium comprising tunicamycin.

Still further, the present invention provides a method of producing a transformed plant comprising a step of transforming a plant cell with said vector.

Advantageous Effects

According to the present invention, GPT gene can be used as a marker for selec ting a genetically modified plant. In addition, since GPT gene can be used as a substit ute for a gene which has resistance to antibiotics or herbicides and may cause a disturb ance in an ecological system, the safety of the genetically modified plants can be impro ved, and therefore it can be advantageously used in an industrial field of genetically mo dified food products.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows callus formation and regeneration of a potato in which leaf fragments of a potato were cultured in a regeneration medium comprising different concentration of tuni camycin.

Fig. 2A shows a schematic diagram of pCAMBIA1301 vector comprising GUS gene f or plant transformation, that is employed in the present invention for the comparison of a co ndition required for transformation. Further, Fig. 2B shows pCAMBIAI 301 -GPT-GUS vect or for plant expression in which hygromycin resistant gene of pCAMBIAI 301 is digested wit h restriction enzymes and GPT gene originating from a potato is ligated to CaMV 35S prom oter so that it can be used as a selection marker.

Fig. 3A shows an electrophoresis result to determine whether or not the transformati on occurred successfully. Specifically, gDNA was extracted from the selected plant and G PT gene of the potato, which had been introduced into the plant genome, was amplified by PCR for the determination. Fig. 3B is a photo of Northern blot analysis showing that GPT gene is expressed in the GPT transformed plant but not in a negative control group.

Fig. 4A shows the expression of GUS in GPT-transformed plants wherein the expres sion of GUS is visualized by GUS staining. PC represents a positive control and NC repre sents a negative control. Fig. 4B is a graph which indicates transformation efficiency betw een GPT and other selection markers.

Fig. 5 is a graph which represents transformation efficiency of potato with different co ncentration of tunicamycin wherein transformation was carried out by using pCAMBIAI 301- GPT-GUS vector.

Fig. 6 is a photo which indicates that secondary selection of a transformant can be c arried out in a medium comprising tunicamycin with a concentration of 0.3-1 ppm after rege nerated plant grows in a medium comprising tunicamycin when transformation experiment was carried out using pCAMBIAI 301 -GPT-GUS vector.

Fig. 7 (A) is the photo of the control plant and the GPT-transformed plant, both h arvested after their cultivation in a greenhouse. Fig. 7 (B) and (C) are the graphs whic h indicate an excellent results in terms of the growth and harvested tuber weight of the potato plant that had been cultivated in a greenhouse.

BEST MODE FOR CARRYING OUT THE INVENTION

In order to achieve the purpose of invention described in the above, the present inve ntion provides a marker consisting of GPT (UDP-Λ/-acetylglucosamine:dolichol phosphate-Λ/ -acetylglucosamine-phosphotransferase) gene, which can be used to selectively determine whether or not a plant is transformed. GPT catalyzes an initial reaction which is necessary for the synthesis of dolichol-p-p- oligosaccharides during dolichol pathway and the glycosylation of Asn-linked glycoproteins. In addition, as a protein present in most of animals and plants, even after it is spread in n ature after being used in a transformed plant GPT would cause no problem of disturbing an ecological system so that the safety of genetically modified plant can be guaranteed.

Because tunicamycin is a strong inhibitor of GPT, when a plant is cultivated in a med ium comprising tunicamycin, GPT of the plant is inhibited by tunicamycin and the plant cann ot survive. Protein N-glycosylation is necessary for plant growth, development, and differe ntiation. According to the present invention, when a plant cell is transformed with a recombi nant plant expression vector comprising GPT encoding gene and the resulting transformed plant cells are propagated in a medium comprising tunicamycin, the plant cells that are tran sformed with the recombinant plant expression vector will overexpress GPT. As a result, a lthough some of the expressed GPT are inhibited by tunicamycin present in the medium, th e remaining GPT is not inhibited by tunicamycin, and therefore the transformed plant can s urvive without inhibition of protein N-glycosylation in the medium comprising tunicamycin. On the other hand, plant cells that are not transformed with the recombinant plant expressio n vector will not express GPT and the tunicamycin comprised in the medium will inhibit the GPT of the plant. As a result, the plant cannot survive in the medium comprising tunicamy cin. Based on such principle, GPT gene can be used as a selection marker to select a tra nsformed plant among non-transformed ones.

Preferably, the above-described GPT gene may consist of a nucleotide sequence th at is represented by SEQ ID NO: 1. Further, variant of this nucleotide sequence is also inc luded in the scope of the present invention. Variant indicates a nucleotide sequence which has a functional characteristic that is similar to that of SEQ ID NO: 1 but has a difference i n nucleotide sequence compared to it. Specifically, GPT gene may comprise a nucleotide sequence which has preferably at least 70%, more preferably at least 80%, still more prefer ably at least 90%, and most preferably at least 95% homology with the nucleotide sequence Of SEQ ID NO: 1.

Further, the present invention provides a recombinant plant expression vector which comprises GPT gene. This vector can be used to see whether or not a plant is transforme d. A method for such selection is the same as those described in the above. The above- described GPT gene may preferably consist of a nucleotide sequence that is represented b y SEQ ID NO: 1. The above-described vector is prepared by inserting tunicamycin-resista nt gene instead of kanamycin- or hygromycin-resistant gene that are commonly used for a v ector for plant expression. Specifically, GPT is inserted in forward direction relative to Ca MV35S promoter and GUS gene is comprised as a reporter gene. Preferably, the vector c an be pCAMBIAI 301 -GPT-GUS vector described in Fig. 2, but is not limited thereto. Any vector which is capable of expressing a target gene can be used.

The term "recombinant" indicates a cell which replicates a heterogeneous nucleotide or expresses said nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. Recombinant cell can express a gene or a gene fragment, th at are not found in natural state of cell, in a form of a sense or antisense. In addition, a rec ombinant cell can express a gene that is found in natural state, provided that said gene is m odified and re-introduced into the cell by an artificial means.

The term "vector" is used herein to refer DNA fragment (s) and nucleotide molecules that are delivered to a cell. Vector can be used for the replication of DNA and be independ ently reproduced in a host cell. The terms "delivery system" and "vector" are often intercha ngeably used. The term "expression vector" means a recombinant DNA molecule comprisi ng a desired coding sequence and other appropriate nucleotide sequences that are essenti al for the expression of the operatively-linked coding sequence in a specific host organism.

A preferred example of a plant expression vector is Ti-plasmid vector which can tran sfer a part of itself, i.e., so-called T-region, to a plant cell when the vector is present in an a ppropriate host such as Agrobacterium tumefaciens. Other types of Ti-plasmid vector (see , EP 0 116 718 B1) are currently used for transferring a hybrid DNA sequence to protoplast s that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a pla nt genome. Especially preferred form of Ti-plasmid vector is a so-called binary vector whic h has been disclosed in EP 0 120 516 B1 and USP No. 4,940,838. Other appropriate vect ors that can be used for introducing the GPT gene of the present invention to a host plant c an be selected from a viral vector such as those originating from a double-stranded plant vir us (e.g., CaMV), a single-stranded plant virus, and Gemini virus, etc., for example a non-co mplete plant viral vector. Use of said vector can be especially advantageous when a plant host cannot be appropriately transformed.

For the plant expression vector according to one embodiment of the present inventio n, a promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, b ut not limited thereto. The term "promoter" means a DNA molecule to which RNA polymer ase binds in order to initiate its transcription, and it corresponds to a DNA region upstream of a structural gene. The term "plant promoter" indicates a promoter which can initiate tran scription in a plant cell. The term "constitutive promoter" indicates a promoter which is acti ve in most of environmental conditions and development states or cell differentiation states. Because the selection of a transformant can be carried out for various plant tissues at var ious stages, the constitutive promoter can be preferred in the present invention. As such, choice of the constitutive promoter is not specifically limited.

For the above-described plant expression vector according to one embodiment of th e present invention, any conventional terminator can be used as a terminator. Example in eludes, nopaline synthase (NOS), rice α-amylase RAmyi A terminator, phaseoline terminat or, and a terminator for octopine gene of Agrobacterium tumefaciens, and the like, but not Ii mited thereto. With respect to the necessity of such terminator, it has been generally known that the presence of such region can increase the efficiency and the certainty of transcripti on in a plant cell. Therefore, the use of a terminator is very much preferred in view of the c ontext of the present invention.

Further, the present invention provides a plant that is transformed with the recombin ant vector of the present invention described in the above. The transformed plant of the pr esent invention expresses GPT. The plant according to one embodiment of the present in vention includes a dicot plant such as potato, Arabidopsis, eggplant, tobacco, pepper, toma to, burdock, crown daisy, lettuce, Chinese bellflower, chard, spinach, sweet potato, celery, c arrot, coriander, parsley, Chinese cabbage, cabbage, leaf mustard, watermelon, melon, cue umber, zucchini, gourd, strawberry, soy bean, mung bean, kidney bean, sweet pea and the like. Preferably, the plant is a potato.

Further, the present invention provides seeds of the above-described plant.

Further, the present invention provides a method of selecting a transformed plant co mprising steps of:

- transforming a plant cell with said vector; and

- selecting a transformed plant in a medium comprising tunicamycin.

The method of selecting a transformed plant according to the present invention com prises a step of transforming a plant cell with the vector of the present invention.

Plant transformation means any method by which DNA is delivered to a plant. Such transformation method does not necessarily have a period for regeneration and/or tissue c ulture. In principle, any transformation method can be used for introducing a hybrid DNA o f the present invention to an appropriate progenitor cells. It can be appropriately selected f rom a calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant MoI. Biol. 8, 363-373), an electroporation me thod for protoplasts (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), a microscopic inje ction method for plant components (Crossway A. et al., 1986, MoI. Gen. Genet. 202, 179-1 85), a particle bombardment method for various plant components (DNA or RNA-coated) (K lein T.M. et al., 1987, Nature 327, 70), or a (non-complete) viral infection method in Agroba cterium tumefaciens mediated gene transfer by plant invasion or transformation of fully ripe ned pollen or microspore (EP 0 301 316), etc. So-called binary vector technique as disclo sed in EP A 120 516 and USP No. 4,940,838 is particularly preferred in the present inventio n.

The "plant cell" that is used for the plant transformation according to the present inve ntion can be any plant cell. The plant cell can be a cultured cell, a cultured tissue, a cultur ed organ, or a whole plant, preferably a cultured cell, a cultured tissue or a cultured organ, and more preferably any form of a cultured cell. Preferably, the above-described plant is a potato.

The "plant tissue" includes either differentiated or undifferentiated plant tissue, includ ing root, stem, leaf, pollen, seed, cancerous tissue and cells having various shape that are used for culture, i.e., single cell, protoplast, bud and callus tissue, but not limited thereto. Plant tissue can be in planta or in a state of organ culture, tissue culture or cell culture.

The method according to the present invention comprises a step of selecting a transf ormed plant cell in a medium comprising tunicamycin. According to the present invention, when a plant is transformed with a recombinant plant expression vector comprising GPT en coding gene and the resulting transformed plant cells are propagated in a medium comprisi ng tunicamycin, the plant cells that are transformed with the recombinant plant expression v ector will overexpress GPT. As a result, although some of the expressed GPT are inhibite d by tunicamycin present in the medium, the remaining GPT is not inhibited by tunicamycin, and therefore the transformed plant can survive without inhibition of protein N-glycosylatio n in the medium comprising tunicamycin. On the other hand, plant cells that are not transf ormed with the recombinant plant expression vector will not express GPT and the tunicamy cin comprised in the medium will inhibit the GPT of the plant. As a result, the plant cannot survive in the medium comprising tunicamycin. Based on such principle, GPT gene can b e used as a selection marker to select a transformed plant among non-transformed ones. According to the method of one embodiment of the present invention, the concentrati on of tunicamycin comprised in the above-described medium is preferably in the range of 0. 05-0.3 ppm. When the concentration of tunicamycin is less than 0.05 ppm, it is difficult to select a transformant from a non-transformant. In addition, when it is more than 0.3 ppm, t he regeneration ratio of the transformed plant tends to decrease.

Still further, the present invention provides a method of producing a transformed plan t comprising steps of:

- transforming a plant cell with the vector of the present invention;

- amplifying the above-obtained transformed plant cell in a medium comprising tunica mycin; and

- regenerating the transformed plant from the above-obtained transformed plant cell. Method of transforming a plant cell is the same as those described in the above, and for a method of regenerating a transformed plant, a method that is publicly well-known in t he pertinent art can be used.

According to the method of one embodiment of the present invention, the concentrati on of tunicamycin comprised in the above-described medium is preferably in the range of 0. 05-0.3 ppm. When the concentration of tunicamycin is less than 0.05 ppm, it is difficult to select a transformant from a non-transformant. In addition, when it is more than 0.3 ppm, t he regeneration ratio of the transformed plant tends to decrease.

The present invention will now be described in greater detail with reference to the foil owing examples. However, it is only to specifically exemplify the present invention and in n o case the scope of the present invention is limited by these examples.

Examples

Example 1 : Determination of resistance of a potato to tunicamvcin at various c oncentrations

In order to determine the resistance of a potato, potato leaves which had been cultiv ated in a culture flask approximately for 15 days were cut, and then cultivated in a plant reg eneration medium (PR medium) which comprises 0.01 mg/£ naphthalenacetic acid, (NAA), 0 .1 Tog/i gibberelin (GA₃), 2.0 mg/t zeatin and 0.01 , 0.05, 0.1 , or 0.3 mgtf tunicamycin. For th e PR medium comprising 0.01 or 0.05 rng/t tunicamycin, it was observed that ten days after the transfer callus was formed at the wound region of the leaf fragments and even a small p lant started to grow after the lapse of four weeks. On the other hand, for the PR medium c omprising higher concentration of tunica mycin, a significant yellowing phenomenon was obs erved after the lapse of one month although some callus formation was found (see, Fig. 1).

Example 2: Preparation of a plant expression vector which expresses GPT gen e

<2-1> Plasmid for plant expression pCAMBIAI 301 -GPT-GUS vector, which is a plasmid vector for transformation of a p otato, was prepared (see, Fig. 2B). Specifically, from pCAMBIAI 301 vector (CAMBIA GP O Box3200, Fig. 2A) which uses hygromycin as a selection marker, hygromycin resistant ge ne was removed by using Xho\ restriction enzyme. After confirming a sequence with PCR synthesis based on the cDNA sequence of a potato that is represented by SEQ ID NO: 1 , G PT gene was ligated to CaMV35S promoter in forward direction. In this case, GPT gene w as ligated to the restriction site of Xho\ and a polymerase chain reaction (PCR) was employ ed to introduce the restriction enzyme site. In detail, in order to obtain expression between CaMV35S promoter and CaMV35S polyA sequence, 5' and 3' terminal restriction enzyme site for Xho\ was artificially introduced to the end region of both primers. PCR was then ca rried out using thus-obtained primer to amplify GPT gene so that it can be introduced into a vector for cloning. For the PCR reaction, denaturation was first carried out at 94 ^°C for 5 mi nutes as a precycling reaction, followed by repeating the cylce thirty times wherein the cycle comprised a sequential treatment including heating at 94 ^°C for 30 seconds, cooling at 60 ⁰C for 30 seconds and heating again at 72⁰C for 45 seconds. As a last reaction step, the mixture was heated at 72⁰C for 10 minutes to terminate the PCR reaction (se, Fig. 2B).

As a result, pCAMBIAI 301 -GPT-GUS plasmid of each vector was obtained, and it w as then introduced into Agrobacterium tumefaciens LBA 4404 for plant transformation. Fur ther, to investigate a basic condition for transformation, pCAMBIAI 301 plasmid of which rep orter gene is GUS was used.

Example 3: Determination of effectiveness of GPT gene as a selection marker

<3-1> pCAMBIAI 301 -GPT-GUS transformation using Agrobacterium Potato leaves were cut and cultivated in a regeneration medium for six hours followe d by co-cultivation with Agrobacterium tumefaciens LBA 4404 which comprises pCAMBIAI 3 01 -GPT-GUS sequence to induce transformation. Thus-obtained transformed leaf fragme nts were cultivated for three days in a callus induction medium (CC medium) which includes 2,4-D (2,4-dichlorophenoxy acetic acid). For this, to MS medium 3% sucrose, 0.8% agar ose, and 2.0 mg/t 2,4-D were added and the pH was adjusted to 5.8 to obtain the callus ind uction medium. Thereafter, the leaf fragments were transferred to a plant regeneration me dium (PR medium) which comprises 0.01 mg/t naphthalenacetic acid, (NAA), 1000 mg/l car benicillin disodium, 2.0 mg/£ zeatin and 0.1 mg/t tunicamycin as a selection marker. Ten da ys after the transfer, callus was formed at the wound region of the leaf fragments and even a small plant started to grow after the lapse of four weeks. After cultivating said small plan t to the level at which it can be isolated, it was transferred to MS basic medium and grown i n the same way as normal plants.

<3-2> Selection of a transformed plant using PCR

In order to see whether or not GPT gene was introduced to the plant by transformati on, PCR reaction was carried out. Specifically, from the leaf fragments of the small plant t hat had been selected from a medium comprising tunicamycin, chromosomal DNA was isol ated. The following primers were respectively used for PCR reaction; forward primer 5'-CC A CAA GTG ACA CAC AGT GAG AAA GAG-3' (SEQ ID NO: 2) and reverse primer 5'-AGC TGA TCT CGT ATC ACA GCT TCA-3¹ (SEQ ID NO: 3), and forward primer 5'-CGT GAA A TC AAA AAA CTC GAC GGC-3 '(SEQ ID NO: 4) and reverse primer 5'-AAG TCC GCA TC T TCA TGA CGA CCA-3' (SEQ ID NO: 5). For the PCR reaction, denaturation was first ca rried out at 94⁰C for 5 minutes as a precycling reaction, followed by repeating the cycle thirt y times wherein the cycle comprised a sequential treatment including heating at 94 ^°C for 30 seconds, cooling at 60⁰C for 30 seconds and heating again at 72 ^°C for 45 seconds. As a last reaction step, the mixture was heated at 72 ^°C for 10 minutes to terminate the PCR reac tion (se, Fig. 3A). As it can be seen from Fig. 3A, for the transformed potato plant of the pr esent invention (#8, 14, 16, 64, 82, 84, 86, 88, 109, 182, 183, and 214), PCR products of b oth GPT gene and GUS genes were identified.

Example 4: Northern blot analysis of GPT gene

Total RNA was extracted from the sample by using phenol/SDS method. 25μg of th e total RNA were separated by electrophoresis using 1 % agarose gel which comprised 2.2 M formalin. Upon completion of the electrophoresis, the RNA bands were transferred to a nylon membrane. For GPT probe, a potato cDNA that had been cloned using forward prim er [5'-CCA CAA GTG ACA CAC AGT GAG AAA GAG-3¹ (SEQ ID NO: 2)] and reverse prim er [5'-AGC TGA TCT CGT ATC ACA GCT TCA-3¹ (SEQ ID NO: 3)] as a template was used for PCR reaction. The nylon membrane was hybridized with digoxigenin (DIG)-labeled pro be using PCR DIG probe synthesis kit (manufactured by Roche Molecular Biochemicals). Hybridization was carried out for 16 hours at 42⁰C using a hybridization buffer solution. Th e membrane was washed twice with 2 X SSC, 0.1 % (w/v) SDS for 5 minutes. Thereafter, t he membrane was again washed with 0.1 X SSC, 0.1 % SDS at 68⁰C for 15 minutes, and th e target DNA was characterized based on DIG luminescence detection kit according to the manufacture's instruction (Roche Molecular Biochemicals). The result is shown in Fig 3B. As it can be seen from Fig. 3B, GPT transcript was confirmed from the transformed plant o f the present invention.

Example 5: Identification of a transformant based on GUS staining

First, by using Agrobacterium tumefaciens LBA 4404 in which pCAMBIA1301 having GUS gene has been introduced as a reporter gene, a condition for achieving transformation was investigated. Two months after Agrobacterium tumefaciens LBA 4404 comprising p CAMBIA1301 plasmid was introduced to a plant, the plant stem grown in a regeneration me dium was subjected to GUS staining and the amount of GUS expression was determined (F ig. 4A). For GUS staining, X-Gluc: 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (1 mM), Na-phosphate (10OmM, adjusted to pH 7.0), and potassium ferricyanide (0.5mM) were mixe d and the reaction was allowed to proceed by incubating the mixture at 37 ^°C overnight. As a result, it was confirmed that GUS was expressed in the transformed potato plant of the p resent invention (Fig. 4A).

In Fig. 4B, transformation efficiency of GPT is compared to that of other selection ma rkers. As it can be seen from Fig. 4B, although the transformation efficiency of GPT is not as high as that of hygromycin (Hyg) or Bastar, it is clear that GPT can be used as a selectio n marker to select a transformed plant.

In order to determine the transformation efficiency with different concentration of tuni camycin, after the potato was transformed with pCAMBIAI 301 -GPT-GUS vector, the conce ntration of tunicamycin comprised in the regeneration medium was adjusted to 0, 0.05, 0.1 , 0.2 or 0.3mg/t and then the transformation efficiency was determined for each concentration . As a result, it was found that the transformation efficiency is favorable at the tunicamycin concentration of 0.1-0.3mg/t In particular, it was found that the transformation efficiency i s more favorable at the tunicamycin concentration of 0.2-0.3mg/ϋ(Fig. 5).

Fig. 6 is a photo which supports that a transformant can be secondarily selected in a medium comprising tunicamycin even with the concentration as low as 0.3-1 ppm after the potato transformation was carried out using pCAMBIAI 301 -GPT-GUS vector and the regen erated plant was grown in the regenerated medium comprising tunicamycin. The regenera ted plant, that is marked with an arrow in Fig. 6, corresponds to a plant which did not surviv e the second selection due to the presence of tunicamycin. Therefore, based on the result s described in the above, it is found that a transformant can be more efficiently selected by t reating it with tunicamycin having different concentration at different stage.

Finally, the growth and the harvest amount of the non-transformant and the GP T-transformed plant were compared to each other after cultivating them in a green hous e (see, Fig. 7). As it can be seen from Fig. 7, it is evident that GPT-transformed plants of the present invention (i.e., GPT8 and GPT14) showed excellent agronomical proper ties including plant growth, harvest amount and the like, compared to the non-transform ant (Desiree cultivar).

Claims

1.

A marker consisting of GPT (UDP-Λ/-acetylglucosamine:dolichol phosphate-Λ/-a cetylglucosamine-phosphotransferase) gene to select a transformed plant.

2.

The marker according to Claim 1 , characterized in that said GPT gene consists of a nucleotide sequence that is represented by SEQ ID NO: 1.

3.

A recombinant plant expression vector comprising GPT gene.

4.

The vector according to Claim 3, characterized in that said vector is used to sele ctively determine the transformation of the plant.

5.

The vector according to Claim 3, characterized in that said vector is pCAMBIA13 01 -GPT-GUS vector described in Fig. 2.

6. A plant which is transformed with the vector of Claim 3.

7. The plant according to Claim 6, characterized in that said plant is a dicot plant.

8. Seeds of the plant described in Claim 6.

9.

A method of selecting a transformed plant comprising steps of:

- transforming a plant cell with the vector according to Claim 3; and - selecting a transformed plant in a medium comprising tunicamycin.

10.

The method according to Claim 9, characterized in that the concentration of tunic amycin comprised in said medium is in the range of 0.05-0.3 ppm.

11.

A method of producing a transformed plant comprising steps of:

- transforming a plant cell with the vector according to Claim 3;

- amplifying thus-obtained transformed plant cell in a medium comprising tunicamy cin; and

- regenerating the transformed plant from thus-obtained transformed plant cell.

12.

The method according to Claim 11 , characterized in that the concentration of tuni camycin comprised in said medium is in the range of 0.05-0.3 ppm.