WO2009035205A1

WO2009035205A1 - TRANSFORMED PLANT EXPRESSING β-SITE APP CLEAVING ENZYME (BACE)

Info

Publication number: WO2009035205A1
Application number: PCT/KR2008/003257
Authority: WO
Inventors: Hyun Soon Kim; Jae Heung Jeon; Jung Won Youm; Hyouk Joung; Young Ho Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-09-13
Filing date: 2008-06-11
Publication date: 2009-03-19
Also published as: KR20090027877A

Abstract

The present invention relates to a transformed plant which expresses BACE gene. More specifically, the present invention relates to a transformed plant which expresses β-secretase gene (BACE), a method for the production of said plant and an oral vaccine composition for preventing dementia which comprises said transformed plant as an effective component.

Description

TRANSFORMED PLANT EXPRESSING β-SITE APP CLEAVING ENZYME (BACE)

Technical Field

[1] The present invention relates to a transformed plant which expresses BACE

(β-secretase or β-site APP cleaving enzyme), an enzyme responsible for producing β- amyloid which causes an Alzheimer type dementia. More specifically, by introducing BACE gene into an Agrobacterium using an expression vector for transformation which comprises BACE gene and co-culturing the transformed Agrobacterium with potato leaves or cotyledonous tissues of tomato, transformed potato and tomato plant, which can express BACE gene in massive amounts, can be obtained. Thus, according to the present invention, a mass production of an oral plant vaccine which is a highly valuable product to be used for prevention or treatment of Alzheimer type dementia can be achieved. Background Art

[2] A vaccine market which is now estimated to be around 10 billion US dollars per year is expected to grow constantly and to reach about 23.8 billion US dollars in year 2012. However, several problems of a current system for developing a vaccine include, expensive manufacturing facility, economic burden for having a freeze facility that is required for transport and storage of vaccine, a difficulty associated with vaccine administration, and public's general dislike feel for an injection, etc. In addition, for the sake of children in undeveloped country wherein a benefit of vaccination is hardly being enjoyed, development of an alternative vaccine is now required more than ever over the world. In 1990, World Health Organization (WHO) set a goal that vaccine development should be made in consideration of human safety and stability to heat, convenient formulation into an oral dose form, and wide variety and easiness of administration method. Among the studies which aim for developing alternative vaccines satisfying such requirements, an oral plant vaccine produced from a transformed crop plant has been acknowledged with its potential and is regarded as a new frontier in vaccine development. Oral plant vaccine, which has an advantage of a marriage between stability of a recombinant vaccine and easiness and efficiency for injection of an oral vaccine, is prepared by a plant that is transformed with an antigen gene by using a plant expression vector, and by carrying out an oral administration of the transformed plant an immunological reaction can be induced. [3] According to the report by International Alzheimer Association, it is expected that the number of patients having certain types of dementia will dramatically increase over next few years in the world. It has been predicted in the report that the number of patients suffering from dementia will be doubled or more every 20 years. That is, although the number is currently around 24 million, it is expected to be 42 million and 81 million in year 2020 and 2040, respectively. Among them, Alzheimer type dementia is presumed to be 60% of the whole dementia cases. Senile dementia that is typified by Alzheimer's disease is a degenerative neuronal disease, which starts as a cognitive disorder and progresses into a long and devastating degenerative disorder which eventually destroys basic human spirit. As such, a passive way of coping such as keeping a patient in an isolate facility is not enough to deal with the social and economic burden associated with the disorder. For such reason, more aggressive approach is required such as the development of an agent which can be used for the prevention or the treatment of the disorder. Meanwhile, pharmaceutical agents that have been already developed or are currently being developed are all an agent for reliving early symptoms rather than curing the disorder itself, i.e., they merely help the function of acetylcholinergic neuronal cells. Taken together the results of the studies that have been carried out until now, it is believed that the cause of Alzheimer's disease is due to the death of neuronal cell which is induced by the accumulation of β-amyloid, a toxic protein, in brain. As such, the most effective way of preventing and treating Alzheimer's disease would be to develop a substance which can fundamentally inhibit the degeneration of neurons by inhibiting the production and toxicity of β-amyloid. According to Korean Patent Registration No. 0546066, a transformed plant cell which expresses tandem repeats of β-amyloid gene and plant produced by the same, both can be used for preparing an oral vaccine composition to prevent dementia in human, have been developed.

[4] β-Amyloid is a metabolite produced by the action of a proteolyitc enzyme which is originated from typelinternal membrane protein called APP (amyloid precursor protein), and it is a peptide consisting of from 39 to 43 amino acids with an extracellular domain and a membrane domain. β-Amyloid is produced by the action of enzymes β-secretase and γ-secretase during an amyloidogenic metabolic process.

[5] According to a recent research study, when β-amyloid is administered as a vaccine composition to a transgenic mouse that has been induced to have Alzheimer type dementia by expressing human β-amyloid, a plaque that had been deposited in the brain of the mouse was disappeared while the behavioral disorder is improved (Schenk et al., Nature 400 (1999), 173-177). Such result supports the possibility of treating dementia, for which no distinct preventive or treating agent is present, based on vaccination.

[6] Inhibition of β-secretase which produces toxic β-amyloid protein is another aspect of prevention of dementia and, once accomplished successfully, is believed to be more economical and efficient than removal of already formed β-amyloid. As such, it is getting increasing attention all over the world now (McConlogue et al., JBC doi/ 10.1074 (2007)). However, not only in Korea but also in any other country there has been a try for expressing human BACE gene in a plant. Furthermore, there has been no report regarding the use of a transformed plant, in which said gene is introduced and expressed as a vaccine for the inhibition of β-secretase enzyme. According to the present invention, however, a plant which can express human BACE gene was developed to achieve said goal and considering ingenuity involved with such research and development and an expected effect of the development, it appears to be a very important result.

[7] As having a complete nutritional composition, potato is one of the four major food crops in the world and is cultivated all over the world except very cold or hot regions. As such, if a potato vaccine is developed as desired by the inventors of the present invention, it can be easily spread all over the world wherein potato is routinely cultivated. Thus, a tremendous need for the potato vaccine will naturally follow. In addition, since a technology for mass production of potato microtuber, which is one of the world's top level technologies with industrially practical use, has been already acquired by the inventors of the present invention, a highly valuable product can be produced in mass amount with combined benefits of a dietary oral vaccine and a potato microtuber.

[8] Meanwhile, tomato has a great taste and flavor and is cultivated and consumed all over the world. In addition, as being possible to be eaten as a raw food that is one of the biggest advantages of a plant oral vaccine, it has long been a subject to study by many researchers. Potato and tomato are both the crops of Solanaceae family and can be easily transformed. In addition, based on an experiment relating to the introduction of other industrially useful gene, it has been already proved that a foreign gene which is introduced in potato or tomato can be stably expressed. Disclosure of Invention Technical Problem [9] The present invention was devised to meet the above-described needs. Specifically, with the ultimate purpose of providing a use as a plant oral vaccine for the prevention and the treatment of Alzheimer type dementia in human, a potato and a tomato plant which can expresss BACE gene is developed by incorporating BACE gene in Agrobacterium using an expression vector plasmid which is used for transformation and comprises BACE gene, and co-culturing the transformed Agrobacterium with potato leaves and cotyledonous tissues of tomato. As a result, a transformed potato and tomato plant, which can express BACE gene in massive amounts, were obtained. Moreover, the expression amount of BACE gene in said transformed plants was increased and a condition for stabilizing the protein was optimized, and as a result the present invention was completed. Technical Solution

[10] In order to solve the above problems, the present invention provides a transformed plant which expresses β-secretase (BACE) gene.

[11] Furthermore, the present invention provides a method for the production of a transformed plant, comprising a step of transforming the plant with a recombinant plant expression vector which includesβ-secretase (BACE) gene.

[12] Still furthermore, the present invention provides an oral vaccine composition for preventing dementia which comprises said transformed plant as an effective component.

Advantageous Effects

[13] The transformed potato and tomato plant of the present invention, which can express β-secretase (BACE) gene to produce β-amyloid responsible for Alzheimer type dementia in human, can be developed as a type of food-based vaccine which currently is a hot issue in the world. Brief Description of the Drawings

[14] Figure 1 shows the BACE gene that is incorporated in the cloning vector.

[15] Figure 2 shows a vector for transformation that is obtained by introducing BACE gene to the vectors for plant transformation including pGBSSP2, pAT and pE8.

[16] Figure 3 A shows the transformed potato plant grown from a callus which has been cultured in a culture medium wherein the callus had been induced by inoculation of Agrobacterium and the microtuber formation from the potato plant, while Figure 3B shows the callus formation in pieces of tomato cotyledonous leaf and culture process of the transformed tomato plant. [17] Figure 4 shows the PCR result of the regenerated plant of the transformed tomato and the regenerated plant of the transformed potato of the present invention.

[18] Figure 5 shows a Northern blot analysis of mRNA of BACE gene that are isolated from the transformed plants selected by PCR determination.

[19] Figure 6 shows a Western blot analysis of BACE protein expression by using ABlO antibody.

[20] Figure 7 shows a nucleotide sequence of BACE gene (SEQ ID NQ 6) of which nucleotide sequence has been modified to increase the expression level of BACE gene and to improve the stabilization of BACE protein in the transformed plants of the present invention. Mode for the Invention

[21] In order to achieve the object of the invention described above, the present invention provides a transformed plant which expresses β-secretase (BACE) gene. The β- secretase (BACE) gene according to the present invention is originated from a human and preferably consists of a nucleotide sequence of SEQ ID NQ 5. Further, variants of the said sequence are within the scope of the present invention. The variants have a different nucleotide sequence but have similar functional characteristics to those of the nucleotide sequence of SEQ ID NQ 5. Specifically, BACE gene may comprise a nucleotide sequence with at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% homology with the nucleotide sequence of SEQ ID NQ 5.

[22] The above-described transformation is characterized in that it is achieved by the incorporation of a vector comprising β-secretase (BACE) gene into the cells of a potato or tomato plant.

[23] Plant transformation means any method by which DNA is delivered to a plant. Such transformation method does not necessarily need a period for regeneration and/or tissue culture. Transformation of plant species is now quite general not only for dicot plants but also for monocot plants. In principle, any transformation method can be used for introducing a hybrid DNA of the present invention to appropriate progenitor cells. The method can be appropriately selected from 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 method for protoplasts (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), a microscopic injection method for plant components (Crossway A. et al., 1986, MoI. Gen. Genet. 202, 179-185), a particle bombardment method for various plant components (DNA or RNA-coated) (Klein T.M. et al., 1987, Nature 327, 70), or a (non-complete) viral infection method in Agrobacterium tumefaciens mediated gene transfer by plant invasion or transformation of fully ripened pollen or microspore (EP 0 301 316), etc. A method preferred in the present invention includes Agrobacterium mediated DNA transfer. In particular, so- called binary vector technique as disclosed in EP A 120 516 and USP No. 4,940,838 can be preferably adopted for the present invention.

[24] The "plant cell" that can be used for the plant transformation in the present invention can be any type of plant cell. It includes a cultured cell, a cultured tissue, a cultured organ or a whole plant, preferably a cultured cell, a cultured tissue or a cultured organ, and more preferably any type of a cultured cell.

[25] The term "plant tissue" can be either differentiated or undifferentiated plant tissue, including 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 plant which can be used in the present invention for the transformation to express the gene of β-secretase ( BACE), which is the enzyme causing Alzheimer type dementia in human, can be food crops including rice, wheat, barley, corn, soy bean, potato, red bean, oat and millet; vegetable crops including Arabidopsis thaUana, Chinese cabbage, radish, hot pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, zucchini, scallion, onion and carrot; special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, wild sesame, peanut and rapseed; fruits including apple, pear, date, peach, kiwi, grape, tangerine, orange, persimmon, plum, apricot and banana; flowers including rose, gladiolus, gerbera, carnation, chrysanthemum, lily, and tulip; and feed crops including rye grass, red clover, orchard grass, alfalfa, tall fescue, and perennial rye grass. Preferably, it is a plant belonging to Solanaceae family, more preferably a potato or a tomato.

[26] The above-described transformation is preferably carried out based on the incorporation of Agrobacterium into plant cells. There is no special limitation for Agrobacterium that can be used for the present invention. However, it is preferably Agrobacterium tumefaciens LB4404. Meanwhile, as to the expression vector which can be used for introducing β-secretase gene into said Agrobacterium, it is preferable to use an expression plasmid consisting of a kanamycin-resistant gene and CaMV 35 promoter, patatin promoter which is specific to potato tuber, or GBSS promoter, but is not limited thereto.

[27] According to a preferred example of the present invention, to produce a potato and a tomato plant which expresses BACE, an expression plasmid for plant transformation comprising BACE gene was introduced into Agrobacterium, potato leaf pieces cultured in vitro and cotyledon pieces of tomato seedlings which were then approximately a week old after the germination were co-cultured with said Agrobacterium, yielding a transformed plant. During the co-culture process with the plant tissue, leaf pieces were scored with a knife and an organ differentiation was induced from the callus that is formed in the scored region. By doing so, the plant was regenerated. The transformed and regenerated individual plant was propagated according to a culture method that is appropriate for each type of plant. Especially for a potato, a massive formation of mi- crotuber is induced under the culture condition comprising a high concentration of sugar such as 9% or more. The expression vector for plant transformation which comprises BACE gene and the Agrobacterium cells wherein said expression vector is incorporated are both within the scope of the present invention. These expression vector and the Agrobacterium cells can be used for various crop plants other than potato and tomato that are tested in the examples of the present invention. In other words, it is clear that there is no specific limitation for a plant cell which can be used for the expression of BACE gene. Preferably, it is a food crop and more preferably is a potato, a tomato or a lettuce, etc. that can be easily cultivated by the people of Korea.

[28] Furthermore, the present invention provides a method for the production of a transformed plant comprising a step of transforming the plant with a recombinant plant expression vector including β-secretase gene (BACE). Preferably, the plant is the one belonging to Solanaceae family, and more preferably a potato or a tomato.

[29] A preferred example of plant expression vector is Ti-plasmid vector which can 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 Ti- plasmid vector (see, EP 0 116 718 B1) are currently used for transferring a hybrid gene to protoplasts that can produce a new plant by appropriately inserting a plant cell or hybrid DNA to a genome of a plant. Especially preferred form of Ti-plasmid vector is a so called binary vector which has been disclosed in EP 0 120 516 Bl and USP No. 4,940,838. Other vector that can be used for introducing the DNA of the present invention to a host plant can be selected from a double- stranded plant virus (e.g., CaMV), a single- stranded plant virus, and a viral vector which can be originated from Gemini virus, etc., for example a non-complete plant viral vector. Use of said vector can be advantageous especially when a plant host cannot be appropriately transformed. Expression vector would comprise at least one selective marker. Said selective marker is a nucleotide sequence having a property based on that it can be selected by a common chemical method. Every gene which can be used for the differentiation of transformed cells from non-transformed cell can be a selective marker. Example includes, a gene resistant to herbicide such as glyphosate and phosphintricin, and a gene resistant to antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol, but not limited thereto.

[30] For the plant expression vector according to one embodiment of the present invention, a promoter can be any of CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter, but not limited thereto. The term "promoter" means a DNA molecule to which RNA polymerase binds in order to initiate its transcription, it corresponds to a DNA region upstream of a structural gene. The term "plant promoter" indicates a promoter which can initiate transcription in a plant cell. The term "constitutive promoter" indicates a promoter which is active in most of environmental conditions and development states or cell differentiation states. Since a transformant can be selected with various mechanisms at various stages, a constitutive promoter can be preferable for the present invention. Therefore, a possibility for choosing a constitutive promoter is not limited herein.

[31] For the above-described terminator, any conventional terminator can be used for the present invention. Example includes, nopaline synthase (NOS), rice α-amylase RAmyl A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens, etc., but are not limited thereto. Regarding the necessity of terminator, it is generally known that such region can increase a reliability and an efficiency of transcription in plant cells. Therefore, the use of terminator is highly preferable in view of the contexts of the present invention.

[32] Finally, the present invention provides an oral vaccine composition for preventing dementia which comprises the above-described transformed plant as an effective component. Preferably, the plant is a potato or a tomato, but not limited thereto.

[33] The above-described composition can be used as a transformed plant itself or as a powder after drying. In addition, it can be used with other food or food ingredients, and can be used appropriately by following a common method. The content of the transformed plant as an effective component is suitably determined depending on the purpose (prevention or therapeutic treatment, or health improvement, etc.). Since the transformed plant as an effective component usually has no safety problem, it can be used without any maximum limit.

[34] Types of health food product which comprises the above-described composition are not specifically limited. Example of the food product to which the transformed plant of the present invention can be incorporated includes, meat, sausage, bread, chocolate, candy, snack, cookie, pizza, ramen and other types of noodles, gum, dairy products including ice cream, various kinds of soup, drink, tea, beverage, alcoholic beverage and vitamin supplement, etc. However, it would be evident to a skilled person in the art that they are not limited to said examples.

[35] The present invention will now be described in greater detail with reference to the following examples. However, it is only to specifically exemplify the present invention and in no case the scope of the present invention is limited by these examples.

[36]

[37] Examples

[38] [Example 1] Plant regeneration based on organ differentiation through callus formation

[39] As a preferred example of the present invention, Solanaceae family plant such as potato and tomato was used as a subject plant and the potato plant tested in the present invention was maintained by tissue culture. Desiree seed potato, which is free of any pathogens and viruses, was germinated and then cut into tiny pieces including growth point, followed by surface sterilization with 70% ethanol. After the rinse with distilled water, the potato pieces were sterilized again in a solution of sodium hypochloride for 10 min, rinsed with distilled water three times and placed in a basic medium wherein sucrose (3%) was added with MS salt to induce stem growth. Once the stems have grown well in an incubator, tissue culture of the potato plant was maintained by sub- culturing every two weeks. Medium for in vitro microtuberization was MS basic medium comprising 90g/L sucrose. Culture condition includes light period for eight hours and culture temperature of 17⁰C. For in vitro microtuberization, lower part of the stems of the potato plant, which had been grown from two to three weeks under said culture condition, was selected for the culture. Approximately two weeks after the culture, the storage stems became to swell and approximately eight weeks later, a mature microtuber having a weight of 1 g or more could be obtained.

[40] Approximately one- week old leaves were cut and used for co-culture with

Agrobacterium for transformation. The medium used for forming callus was MS medium to which 3% sucrose, 8% agar and 2.0 mg/L 2,4-D (2,4-dichlorophenoxy acetic acid) were added. The medium was adjusted to have pH 5.8. The transformed leaf pieces were cultured for two days in medium for inducing callus formation which comprises 2,4-D, and then transferred to a medium for regeneration which comprises 0.01 mg/L NAA (naphthaleneacetic acid), 0.1 mg/L gibberellin (GA₃)and 2.0 mg/L zeatin. One week after the transfer, calluses started to form at the cut area of the leaf pieces, and four weeks later small plants started to appear. After culturing the plants to the stage that they can be isolated, the plants were transferred to MS basic medium wherein they were allowed to grow same as normal plants to induce formation of a mi- crotuber. Tomato seeds (Dotaerang, a tomato variety for general cultivation) were first treated with 70% ethanol for surface sterilization followed by rinsing with distilled water. Then, the seeds were sterilized again in a 10% solution of sodium hypochloride for 10 min, rinsed with distilled water three times and placed in a basic medium wherein sucrose (3%) was added with MS salt to induce germination. About one week after the germination, newly-formed cotyledonous leaves were cut and used for co- culture with Agrobacterium for transformation. The medium used for forming callus was MS medium to which 3% sucrose, 8% agar and 1.0 mg/L of zeatin were added. The medium was adjusted to have pH 5.8. The transformed cotyledonous leaf pieces were cultured for two days in medium for inducing callus formation, and then transferred to a medium for regeneration comprising 2.0 mg/L zeatin. One week after the transfer, calluses started to form at the cut area of the leaf pieces, and four weeks later small plants started to appear (see, Figure 3).

[41] [Example 2] Introduction of BACE gene to the leaves of potato and tomato

[42] In order to introduce BACE gene (SEQ ID NQ 5, as confirmed in Figure 5) to the leaves of potato and tomato, potato leaves that have been cultured less than seven days as described in Example 1 and approximately one hundred tomato cotyledonous leaves that had been obtained about one week after the germination were selected and dipped in a culture solution comprising Agrobacterium tumefaciens LBA 4404 for ten minutes. Moisture was completely removed by drying with a sterilized paper. The potato leaf pieces were then placed in a co-culture medium which comprises 2.0 mg/L 2,4-D while the tomato cotyledonous leaf pieces were placed in a co-culture medium which comprises 1.0 mg/L zeatin. The leaf pieces were culture for two days, respectively. According to this process, callus formation was more easily induced at cut region of the leaf pieces. In this case, antibiotics such as kanamycin should not be added to increase a chance for Agrobacterium to incorporate to the plant genome. When the callus was about to form in the leaf pieces near cut region, the potato leaf pieces were transferred to MS agar medium which comprises 0.01 mg/L NAA, 0.1 mg/ L GA₃ and 2.0 mg/L zeatin while the tomato cotyledonous leaf pieces were transferred to MS medium for regeneration which comprises 2.0 mg/L zeatin. At the same time, 100 mg/L of kanamycin was added thereto to give a pressure for selection by which only the transformed plants can be regenerated. Meanwhile, to remove any Agrobacterium that had been stuck to the leaf pieces or left free without being incorporated in the plants, 1,000 mg/L of carbenicillin was added to the culture medium. While being regenerated, the transformed small plants were transferred to a new medium every two weeks. For such transformational processes, plasmids including pATBACE, pGBSSP2BACE and pE8BACE, which have been obtained by linking human BACE gene to patatin promoter that is a promoter specific to potato tuber and Granule Bound Starch Synthase (GBSS) promoter, were used. In addition, only for the tomato, a vector that is linked to E8 promoter, which is a fruit-ripening specific promoter, was used (see, Figure 2). From the vector for plant expression in which patatin promoter and GBSSP2 promoter were introduced, GUS gene was removed with restriction enzymes of Kpnland Sac I followed by insertion of BACE gene (SEQ ID NQ 5) to yield the vectors of pATBACE and pGBSSP2BACE, respectively. For the vector comprising E8 promoter, BamHI and Kpnl were used as a restriction enzyme. These vectors carry kanamycin-resistant nptll gene that was employed as a selection marker. Subsequently, plasmid DNAs comprising each of said vectors were introduced to Agrobacterium tumefaciens LBA 4404 and used for plant transformation of the present invention.

[43] The resulting transformed leaf pieces were subcultured every two weeks by using the same selection medium. Eight weeks after starting the culture, about twenty small plants were obtained for the each vector tested. Compared to a normal plant obtained by in vitro culture, the plants looked normal without any odd morphology. According to the subsequent cultivation of the potato plants after they had been transferred to a medium for microtuberization, even a normal potato microtuber was obtained. The in vitro small plants of the tomato were transferred to a pot and allowed to grow. As a result, they were all grown into a normal individual plant. Taken together, it was confirmed that BACE gene of the present invention was stably incorporated into the potato and tomato plants, which were then grown into a normal plant. [44] [Example 3] Confirmation of BACE gene incorporation by PCR analysis

[45] In order to confirm the incorporation of BACE gene of the present invention in the plant, a PCR (polymerase chain reaction) was carried out. By using a selection medium comprising kanamycin, small plants that could grow in the medium were primarily selected. Then, PCR was carried out to identify the presence of BACE gene in the genomic DNA of the selected plants. Specifically, genomic DNA was first isolated from the leaf pieces of the small plant based on a medium comprising kanamycin, and PCR was carried out by using a primer of BACE gene and NPTII. With a forward primer (5'-ATG ATT GAA CAA GAT GGA-3'; SEQ ID NQ 1) and a reverse primer (5'-TCA GAA GAA CTC GTC AAG-3'; SEQ ID NQ 2) of NPT II, the genomic DNA was denatured for 2 min at 94⁰C as a precycling reaction. Then, a heating and cooling cycle including 45sec at 94⁰C, 45sec at 57⁰C, and 1 min at 72⁰C in said order was repeated twenty five times and as a last step the reaction was carried out at 72⁰C for one minute to complete the reaction. According to an agarose gel analysis, DNA band corresponding to NPTII gene (0.75kb) was confirmed. Meanwhile, with a forward primer (5'-ATG ACC CAG CAC GGC ATC CGG CTG CCC CTG-3'; SEQ ID NQ 3) and a reverse primer (5'-TCA ATA GGC TAT GGT CAT GAG GGT TGA CTC ATC-3'; SEQ ID NQ 4) of BACE, the genomic DNA was denatured for 2 min at 94⁰C as a precycling reaction. Then, a heating and cooling cycle including 45sec at 94⁰C, 50sec at 55⁰C, and 1 min at 72⁰C in said order was repeated twenty five times and as a last step the reaction was carried out at 72⁰C for five minutes to complete the reaction. According to an agarose gel analysis, DNA band corresponding to BACE gene (1.3kb) was confirmed. Taken together the above results, it was confirmed that BACE gene and NPTII gene as a selection marker are present in the transformed potato and tomato plants of the present invention (Figure 4). Three and six plants were found to have BACE gene incorporated therein for p ATBACE (Figure 4A) and pGBSSBACE (Figure 4B), respectively. Moreover, for pE8BACE plant of which expression is under control of tomato-ripening specific promoter E8, individual plants confirmed with the presence of NPTII gene (Figure 4C) and BACE 850bp fragment (Figure 4D) therein were successfully selected.

[46] [Example 4] Analysis of BACE transcripts from the transformed potato and tomato plants

[47] According to this experiment, by first confirming the RNA expression in the transformed potato and tomato plants of the present invention, a more precise selection of transformed plant can be achieved. Considering that the vectors used in the present invention are specific to either potato tuber or ripening of tomato fruit, transcripts were identified from the regions (e.g., plant organs) in which said specific features are expressed. For potato, total RNA was extracted from 1 g of newly formed microtuber. For tomato, Ig of fully-ripen red pulp was used for RNA analysis. After quantification, 30μg of purified RNA was applied to 1% agarose gel comprising 19.8% formaldehyde. Upon the completion of electrophoresis, RNAs contained in the gel were transferred to a nylon membrane, without any special pre-treatment. To fix the transferred RNA on the nylon membrane, UV light was illuminated twice to the membrane (1200xμJ/cnf). In order to visually identify the ZJACE- specific RNA band from the above nylon membrane, following treatments were carried out. First, a probe tagged with DIG label was linked to the nylon membrane at 5O⁰C for 16 hours, and then ZJA CE-specific RNA band was visualized by using a diagnostic kit which can detect DIG. As a result of testing many plants, one individual plant showing the highest expression level was selected for each individual vectors. That is, plant Number 9 for the transformed potato comprising pATBACΕ vector (Figure 5A) and plant Number 40 for the transformed potato comprising pGBSSBACΕ vector (Figure 5B) were selected. In addition, plant Number 3 for the transformed tomato comprising pΕ8BACΕ vector was also finally selected (Figure 5C).

[48] [Example 5] Analysis of beta-secretase protein expression

[49] BACE protein that had been expressed in the transformed potato and tomato plants of the present invention was analyzed by Western blot. First, to confirm the condition for detection of BACE protein, Western analysis was carried out for pGBSSBACE plant which had been previously selected. As a result, a specific band (-5OkDa) that had not been observed for non-transformed control potato plant was observed (Figure 6A). From the tuber of an individual transformed potato plant and well-ripen fruits of an individual transformed tomato plant, proteins were extracted, respectively. Extraction buffer used was a mixture of PBS buffer (pH 7.2), 10 mM EDTA, 1 mM proteinase inhibitor cocktail, 0.1% Triton X-100, and 5 mM β-mercaptoethanol, which was added in a volume half the weight of the sample. All of the extraction processes were carried out quickly at about 4⁰C. For all of the individual transformed plants tested, a specific band (~50kDa, corresponding to β-secretase potato tuber protein), that had not been observed for non-transformed control potato plant, was observed.

Claims

[1] Transformed plant which expresses β-secretase (BACE) gene.

[2] The transformed plant according to Claim 1, characterized in that said β- secretase gene consists of a nucleotide sequence of SEQ ID NQ 5.

[3] The transformed plant according to Claim 1, characterized in that said plant is either a potato or a tomato.

[4] The transformed plant according to Claim 1, characterized in that said transformation is achieved by the introduction of a vector comprising β-secretase (

BACE) gene to the cells of said plant. [5] A method of producing a transformed plant comprising a step of transforming the plant with a recombinant plant expression vector which comprises β- secretase (BACE) gene. [6] The method according to Claim 5, characterized in that said plant is either a potato or a tomato. [7] An oral vaccine composition for preventing dementia which comprises the transformed plant of Claim 1 as an effective component. [8] The oral vaccine composition according to Claim 7, characterized in that said plant is either a potato or a tomato.