KR100920330B1 - Transgenic lettuce plants producing increased tocopherol content - Google Patents

Abstract

The present invention relates to a transformed lettuce with an increased tocopherol content, more specifically, TC / VTE1 (tocopherol cyclase) derived from Arabidopsis thaliana involved in the synthesis of tocopherol, consisting of the amino acid sequence represented by SEQ ID NO: 4 The present invention relates to a transformed lettuce characterized by an increase in tocopherol content by transformation with a recombinant vector comprising a gene encoding a protein, and a method for increasing the tocopherol content by overexpressing the gene in lettuce.

Lettuce, tocopherol, transformation, gene, TC / VTE1

Description

Transgenic lettuce plants producing increased tocopherol content

The present invention relates to a transgenic lettuce with increased tocopherol content.

Tocopherol, known as vitamin E, is synthesized through photosynthetic organisms as a lipophilic antioxidant (Traber, MG and Sies, H. (1996) Annu. Rev. Nutr. 16, 321-347; Grusak, MA (1999) Trends Plant Sci 4, 164-166). These tocopherols are divided into structurally closely related compounds α-, β-, γ-, and δ-tocopherol.

The biological activities of these four tocopherol isomers vary according to the position and number of the methyl groups of the chromatin ring and the arrangement of the asymmetric carbons of the side chains (Hirschberg, J. (1999) Curr. Opin. Biotech. 10, 186-191 ).

Among them, α-tocopherol is the lipophilic antioxidant with the highest biological activity against animals including humans. In particular, it is estimated that tocopherol contributes to the maintenance and maintenance of biological membranes by preventing oxidation of reactive oxygen species of unsaturated fatty acids constituting membrane fats.

Recent studies have shown that regular use of relatively high levels of tocopherol can inhibit the progression of atherosclerosis. In addition, the physiological characteristics and effects of tocopherol have been examined in delaying acquired damage due to diabetes, reducing the risk of developing cataracts, reducing oxidative stress in smokers, anticancer effects, and anti-skin damage effects.

Such tocopherols are abundant in seed oils of many vegetable oils, in particular soybeans, wheat, corn, rice, cotton, lucene and nuts. It is also contained in fruits and vegetables, such as raspberries, beans, peas, fennel, peppers, and the like.

Tocopherols are biosynthesized from precursors derived from two pathways in the body of the plant and photosynthetic microorganisms (FIG. 1). The methylethylthritol phosphate pathway synthesizes phytyldiphosphate to form the side chains of tocopherols. The shikimate pathway also produces homogentisic acid and provides it to the aromatic ring of tocopherol. The first step in this tocopherol biosynthesis process is the prenylation process of homogentisic acid with phytyldiphosphate to produce 2-methyl-6-phytyl-benzoquinol (MPBQ) (Savidge, B., et al . (2002) Plant Physiol. 129 , 321-332). The total content of tocopherol produced at this time is determined from the substrate specificity and activity of homogentisate phytyltransferase (HPT / VTE2) and tocopherol cyclase (TC / VTE1) and two methyltransferase enzymes (VTE3 and VTE4) (Figure 1). Tocopherol cyclase activity induces the formation of δ-tocopherol from MPBQ. In contrast, methylation by MPBQ methyltransferase (MPBQMT / VTE3) induces the formation of 2,3-dimethyl-5-phytylbenzoquinol (DMPBQ), followed by tocopherol cyclase activity leading to the formation of γ-tocopherol. Finally, γ-tocopherol methyltransferase (γ-TMT / VTE4) forms ring methylation at the R ₃ position, transforming γ and δ isoforms into α and β isoforms, respectively (DellaPenna, D. and Last, RL (2006)). Physiol Plantarum. 126, 356-368; Della Penna, D. and Pogson, BJ (2006) Annu. Rev. Plant Biol. 57, 711-738).

At present, there are reports of Arabidopsis thaliana , which is used to transform genes of various enzymes involved in tocopherol biosynthesis and control their content. For example, expression of the HPT / VTE2 and TC / VTE1 genes from Arabidopsis has increased tocopherol content by 4.4 and 7 fold, respectively, in Arabidopsis leaves (Collakova, E. and Della Penna, D. (2003)). Plant Physiol. 131, 632-642; Kanwischer, M., Porfirova, S., Bergmuller, E. and Dormann, P. (2005) Plant Physiol. 137, 713-723). In order to find out the possibility of increasing the content of tocopherol in foliar crops through metabolic engineering, the present invention overexpressed homogentisate phytyltransferase ( HPT / VTE2 ) and tocopherol cyclase ( TC / VTE1 ) genes Transformed lettuce ( Lactuca sativa L. cv. Chungchima) was prepared, and the composition and content of tocopherol were analyzed.

The present invention has been made in accordance with the above requirements, the present invention is transformed with a vector containing a gene encoding a TC / VTE1 protein derived from Arabidopsis involved in the synthesis of tocopherol transformed lettuce with increased tocopherol content To provide.

In order to solve the above problems, the present invention comprises a gene encoding TC / VTE1 (tocopherol cyclase) protein derived from Arabidopsis thaliana involved in the synthesis of tocopherol, consisting of the amino acid sequence represented by SEQ ID NO: 4 It is to provide a transgenic lettuce characterized in that the tocopherol content is increased by transformation with a recombinant vector.

The present invention also provides a method of increasing the tocopherol content by overexpressing the gene in lettuce.

The present invention can be used in food engineering applications due to the antioxidant activity of tocopherol by providing a transgenic lettuce with increased tocopherol content.

The range of HPT / VTE2 and TC / VTE1 proteins according to the present invention includes proteins having amino acid sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively, isolated from tobacco and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 2 as a result of the addition, substitution, or deletion of the amino acid Is 95% or more of sequence homology, and refers to a protein that exhibits substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 2. "Substantially homogeneous physiological activity" refers to the activity involved in tocopherol synthesis in plants.

The present invention also provides genes encoding the HPT / VTE2 and TC / VTE1 proteins. Genes of the invention include both genomic DNA and cDNA encoding HPT / VTE2 and TC / VTE1 proteins. Preferably, the gene of the present invention may include a nucleotide sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention also provides a recombinant vector comprising the gene according to the present invention. The recombinant vector is preferably a recombinant plant expression vector.

The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term “vector” is used to refer to a DNA fragment (s), a nucleic acid molecule, that is delivered into a cell. Vectors can replicate DNA and be reproduced independently in host cells. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc., For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin (phosphinothricin), kanamycin, G418, bleomycin, hygromycin, chloramphenicol There are antibiotic resistance genes such as, but not limited to.

In the plant expression vector according to an embodiment of the present invention, the promoter may be, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS or histone promoter. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.

The terminator may be a conventional terminator, and examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agrobacterium tumefaciens (ocrobacterium tumefaciens) Terminator of the Fine (Octopine) gene, etc., but is not limited thereto. With regard to the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.

The present invention also provides a plant transformed with the recombinant vector according to the present invention. Plant transformation refers to any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both dicotyledonous plants as well as monocotyledonous plants. In principle, any transformation method can be used to introduce hybrid DNA according to the invention into suitable progenitor cells. Method is calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumulopasis by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. Preferred methods according to the invention include Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

The "plant cells" used for plant transformation may be any plant cells. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to the tissues of differentiated or undifferentiated plants, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

The present invention also provides a method of participating in tocopherol synthesis by expressing the HPT / VTE2 and TC / VTE1 genes according to the present invention in plants.

As a method for expression of the HPT / VTE2 and TC / VTE1 genes in plants is within the HPT / VTE2 and TC / VTE1 each plant containing the gene respectively, and a plant or that do not contain the HPT / VTE2 and TC / VTE1 gene HPT / This can be done by introducing the VTE2 and TC / VTE1 genes respectively. A method for introducing the HPT / VTE2 and TC / VTE1 gene into a plant is a method of using the HPT / VTE2 and TC / VTE1 gene subject to the control of the promoter using the expression vectors each containing the transgenic plant. The promoter is not particularly limited as long as it can express an insertion gene in a plant. Examples of such promoters include, but are not limited to, 35S RNA and 19S RNA promoters of CaMV; Full length transcriptional promoters derived from Peak Water Mosaic Virus (FMV) and Coat protein promoters of TMV. In addition, the ubiquitin promoter can be used to express the HPT / VTE2 and TC / VTE1 genes in monocotyledonous plants or woody plants.

Plants that may be involved in tocopherol synthesis of plants by the method of the present invention include food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including lygras, redclover, orchardgrass, alphaalpha, tolskew, perennial licegrass and the like.

The present invention also provides a transgenic plant in which a change is produced in the tocopherol content produced by the above method.

Plants in which a change in the tocopherol content according to the present invention is produced can be obtained through the sexual propagation method or the asexual propagation method which is a conventional method in the art. More specifically, the plant of the present invention can be obtained through the oily breeding process of producing seeds through the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant vector comprising the HPT / VTE2 and TC / VTE1 gene according to the present invention can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. have. That is, explants of plants transformed with recombinant vectors containing HPT / VTE2 and TC / VTE1 genes are implanted in a suitable medium known in the art and then cultured under appropriate conditions to induce the formation of callus, and shoots are formed. When incubated, transfer to the hormone-free medium. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. By inducing roots and transplanting them into the soil, the plants can be obtained with a change in tocopherol content. In the present invention, the transformed plant may include not only the whole plant, but also tissues, cells, or seeds obtainable therefrom.

Tocopherol, also known as vitamin E, is a fat-soluble antioxidant that is synthesized by plants and photosynthetic microorganisms. Tocopherols prevent the chloroplasts of higher plants from being damaged by photooxidation and are an essential component of a human balanced diet. Tocopherols come in four major forms: alpha, beta, gamma, and delta (α, β, γ, δ). Of the four types of tocopherols present in nature, the alpha tocopherol activity is 100%, which corresponds to 50% of beta tocopherol, 10% of gamma tocopherol, and 3% of delta tocopherol.

In the present invention, transgenic lettuce ( Lactuca sativa L. cv. Chungchima ) overexpressing the Arabidopsis homogentisate phytyltransferase ( HPT / VTE2 ) and tocopherol cyclase ( TC / VTE1 ) genes were prepared, and the composition and content of tocopherol were analyzed.

The genes selected above were selected for the purpose of increasing the content of tocopherol by increasing the overall flux towards the tocopherol biosynthetic pathway. In addition, two enzymes, HPT / VTE2 and TC / VTE1, have been identified as the major limiting factors involved in the synthesis of tocopherols in the leaves of Arabidopsis (Fig. 1). On the other hand, MPBQMT / VTE3, which converts MPBQ to DMPBQ, is not considered to be a limiting factor given that most of the tocopherols found in Arabidopsis and lettuce are the more effective antioxidants gamma and alpha tocopherols than delta and betatocopherol. MPBQMT / VTE3 was therefore excluded as a target for increasing the tocopherol content of lettuce through metabolic engineering. γ-TMT / VTE4 was also excluded from the target on the basis of reports that overexpression in Arabidopsis, lettuce, and soybeans contributed little to the increase in total tocopherol content.

HPT / VTE2 and TC / VTE1 genes were transformed into lettuce using Agrobacterium, respectively, and two transformants were obtained through genomic DNA PCR analysis (FIG. 3). In addition, the real time RT-PCR analysis showed that the amount of transcript of HPT / VTE2 and TC / VTE1 expressed in these transformants was greater than that expressed in wild tyep. Reverse phase HPLC analysis showed that the total tocopherol content of the transformed lettuce was 2.7-fold and 2-fold higher in the leaves of HPT / VTE2 transformants than in the wild-type lettuce, and also 2-fold higher in the leaves of TC / VTE1 transformants. It was measured (FIG. 4; Table 1). The increase in total tocopherol content in both of these transformants was mainly due to the increase in gamma-tocopherol content. On the other hand, beta-tocopherol was not detected in all transformants and wild type lettuce crops.

Artificially manipulating the metabolic pathways in a plant makes it easy to make changes in other metabolic pathways around it. In this context, the chlorophyll content of transgenic lettuce was measured. This is because phytyl-DP, a substrate of HPT / VTE2 enzyme in tocopherol biosynthesis, is also commonly used in chlorophyll biosynthesis. As a result, the content of chlorophyll in the HPT / VTE2 transformant was noticeably reduced by 20% (P <0.01) (Fig. 5). This phenomenon is thought to be due to competition for phytyl-DP between chlorophyll biosynthetic enzymes present in the transformant and the overexpressed HPT / VTE2 enzyme. That is, the flux balance of phytyl-DP shifted toward tocopherol biosynthesis as HPT / VTE2 enzyme, which is a limiting factor in tocopherol biosynthesis, increased in the transformants. On the other hand, in the TC / VTE1 transformants, the chlorophyll content can be seen to increase significantly up to 35% (P <0.001). This is thought to be due to the increase in the amount of phyty-DP that leads to chlorophyll synthesis because only a limited amount of phytyl-DP accumulated by TC / VTE1 is consumed by HPT / VTE2, which is limited in lettuce. .

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Plants and Growth Conditions

Lettuce ( Lactuca sativa L. cv. Chungchima) seed surfaces were sterilized in 70% ethanol for 30 seconds and then treated with 1.0% sodium hypochlorite solution for 15 minutes. Then seeded in MS medium (Murashige, T. and Skoog, F. (1962) Physiol. Plant 15, 473-497) to which 3% (w / v) sucrose and 0.6% (w / v) phytoagar were added. It was. At this time, the pH of the medium was adjusted to 5.8. Seeds were sprouted at 25 ° C., constant photoperiod (16 h light (50 μmol / m ² / s) / 8 h dark).

<Example 2; Construction of Recombinant Plasmids for Transformation of Lettuce>

Total RNA was isolated from young shoots of 2 week old Arabidopsis RNeasy Plant Mini Kit (Qiagen). From this, cDNA of Arabidopsis homogentisate phytyltransferase ( HPT / VTE2 ) was prepared using RT-PCR. The first cDNA strand was synthesized using SuperScript ^™ III RNase H- reverse transcriptase (Invitrogen) and primer (AtHPT-RT, 5'-TCACTTCAAAAAAGGTAACAGCAAGTA-3 ') (SEQ ID NO: 5). The second PCR product contains a set of gene specific primers (AtHPT-L, 5'-CATGCCATGGAGTCTCTGCTCTCTAGTTCT-3 '(SEQ ID NO: 6) and AtHPT-R, 5'-GGGTCACCTCACTTCAAAAAAGGTAACAGC-3' (SEQ ID NO: 7)) for MgSO ₄ and pfu DNA. The reaction was carried out with the polymerase. PCR conditions are as follows. Initial denaturation 95 ° C: 1.5 minutes / 94 ° C: 2 minutes, 60 ° C: 30 seconds, 72 ° C: 2.3 minutes, 40 cycles. The PCR product of HPT / VTE2 thus produced was cut into NcoI and BstEII, and then inserted into pCAMBIA1301 vector. Arabidopsis tocopherol cyclase (TC / VTE1) cDNA was obtained by the above method using AtTC-RT primer (5'-TTACAGACCCGGTGGCTTGAAGAAAGG-3 '(SEQ ID NO: 8)). The gene specific primers used at this stage are At TC-L (5'-CATGCCATGGAGATACGGAGCTTGATTGTT-3 ') (SEQ ID NO: 9) and At TC-R (5'-GGGTCACCTTACAGACCCGGTGGCTTGAA-3' (SEQ ID NO: 10)) The initial denaturation is 95 ° C: 1.5 minutes / 94 ° C: 2 minutes, 60 ° C: 30 seconds, 72 ° C: 2.9 minutes, and 50 cycles.

<Example 3; Transformation of Lettuce Using Agrobacterium>

A single colony of Agrobacterium LBA4404 with either pCAMBIA1301- AtHPT / VTE2 or pCAMBIA1301- AtTC / VTE1 was incubated in a YEP liquid medium containing rifampicin and kanamycin until OD ₆₀₀ was 1.1-1.6. The culture was then centrifuged at 5,000 × g for 20 minutes and the pellet was again taken up in liquid MS medium. It was diluted 10 times with liquid MS medium. After sowing, six to seven days old lettuce cotyledon (cotyledons) were wound three times in the direction perpendicular to the axial surface and inoculated in 1/10 dilution of bacteria for 5 minutes (Wroblewski, T., Tomczak, A. and Michelmore). , R. (2005) Plant Biotechnol. J. 3, 259-273). Drain the cotyledon with a sterile filter and wash it in MRS medium (MS basal salts with vitamins, 3% sucrose, 0.1 mg / L NAA, 0.1 mg / L BAP, pH 5.8, 0.6% phytoagar) Incubated for two days (Kim, JH and Botella, JR (2004) Plant Cell Tiss. Org. 78, 69-73; Kim, B. et al . (2004) J. Kor. Soc. Hort. Sci. 45, 16 -20). Two days later, Agrobacterium treated cotyledon was transferred to MSR medium containing 10 mg / l hygromycin and 300 mg / l cefotaxime. Two weeks later, the cotyledon was transferred to fresh MRS medium supplemented with hygromycin and cefotaxime. Redifferentiated individuals from these sections were transferred to fresh MRS medium containing hygromycin and cefotaxime. The surviving individuals were cultured until roots were formed in MSO medium.

<Example 4: T-DNA insertion check>

PCR reactions to select HPT / VTE2 transgenic subjects were carried out using a gene-specific primer set ( At HPT-M, 5'-GTTTCTGATATATCTCCTTTA-3 '(SEQ ID NO: 11) and pCAM-NosPolyA, 5'-CATGCTTAACGTAATTCAAC-3' (SEQ ID NO: 12)) was used to make a 20 μl reaction solution containing 50 ng of chromosomal DNA. PCR conditions were carried out in the initial denaturation 95 ℃: 4 minutes / 94 ℃: 30 minutes, 55 ℃: 30 seconds, 72 ℃: 1 minutes, 35 cycles. Primer sets for selecting TC / VTE1 transgenic individuals were At TC-L, 5'-CATGCCATGGAGATACGGAGCTTGATTGTT-3 '(SEQ ID NO: 13) and At TC-Rev3, 5'-GAACCCTTCGGACACTCTTCTGTTA-3' (SEQ ID NO: 14). The conditions are as described above.

Example 5 HPLC Analysis for Tocopherol Quantitation

To perform tocopherol quantification of lettuce leaves, the extracts were extracted from 80 mg of frozen leaves using methanol. The extract was filtered using 0.45 μm PVDF, 4 mm syringe filter, and placed in autosampler tubes. Tocopherol isocratic reverse-phase HPLC (58% [v / v] acetonitrile and 42% [v / v] methanol containing 7%-) under conditions of 20 μl injection rate, 1.5 mℓ min ^- 1 flow rate and 20 minutes run time). isopropanol). HPLC analysis was performed using a fluorometer (Shimadzu, RF-10AXL, Japan) and a Phenomenex Luna C18 column (25 cm ⅹ 4.6 mm, 5 μm). At this time, the excitation of the sample was observed at 298 nm and emission at 328 nm. Peaks of α-, β-, γ-, and δ-tocopherols were distinguished by comparison with retention times of standard peaks. Tocopherol concentrations and components were calculated based on the respective standard curves. Tocopherol extraction and HPLC analysis were performed four times, respectively.

Example 6 Chlorophyll Determination

Chlorophyll content of transformants and wild type lettuce leaves was measured spectrophotometrically (Arnon, D. I. (1949) Plant Physiol. 24, 1-15). Extraction and quantification of chlorophyll were performed three times each.

Table 1. Tocopherol content and composition of HPT / VTE2 and TC / VTE1 transformants

1 is a diagram showing the tocopherol biosynthetic pathway of Arabidopsis;

Figure 2 is a diagram showing the transformed lettuce produced by transformation with Agrobacterium ,

FIG. 3 is a diagram confirming whether a foreign gene derived from Arabidopsis herpes is inserted into the genomes of the lettuce HPT / VTE2 and TC / VTE1 transformants through genomic DNA PCR analysis.

4 is a diagram measuring the tocopherol content of wild type lettuce and HPT / VTE2 and TC / VTE1 transformed lettuce,

Figure 5 is a measure of the chlorophyll content of wild type lettuce and HPT / VTE2 and TC / VTE1 transformed lettuce.

<110> Seoul National University Industry Foundation <120> Transgenic lettuce plants producing increased tocopherol content <160> 14 <170> KopatentIn 1.71 <210> 1 <211> 1182 <212> DNA <213> Arabidopsis thaliana <400> 1 atggagtctc tgctctctag ttcttctctt gtttccgctg ctggtgggtt ttgttggaag 60 aagcagaatc taaagctcca ctctttatca gaaatccgag ttctgcgttg tgattcgagt 120 aaagttgtcg caaaaccgaa gtttaggaac aatcttgtta ggcctgatgg tcaaggatct 180 tcattgttgt tgtatccaaa acataagtcg agatttcggg ttaatgccac tgcgggtcag 240 cctgaggctt tcgactcgaa tagcaaacag aagtctttta gagactcgtt agatgcgttt 300 tacaggtttt ctaggcctca tacagttatt ggcacagtgc ttagcatttt atctgtatct 360 ttcttagcag tagagaaggt ttctgatata tctcctttac ttttcactgg catcttggag 420 gctgttgttg cagctctcat gatgaacatt tacatagttg ggctaaatca gttgtctgat 480 gttgaaatag ataaggttaa caagccctat cttccattgg catcaggaga atattctgtt 540 aacaccggca ttgcaatagt agcttccttc tccatcatga gtttctggct tgggtggatt 600 gttggttcat ggccattgtt ctgggctctt tttgtgagtt tcatgctcgg tactgcatac 660 tctatcaatt tgccactttt acggtggaaa agatttgcat tggttgcagc aatgtgtatc 720 ctcgctgtcc gagctattat tgttcaaatc gccttttatc tacatattca gacacatgtg 780 tttggaagac caatcttgtt cactaggcct cttattttcg ccactgcgtt tatgagcttt 840 ttctctgtcg ttattgcatt gtttaaggat atacctgata tcgaagggga taagatattc 900 ggaatccgat cattctctgt aactctgggt cagaaacggg tgttttggac atgtgttaca 960 ctacttcaaa tggcttacgc tgttgcaatt ctagttggag ccacatctcc attcatatgg 1020 agcaaagtca tctcggttgt gggtcatgtt atactcgcaa caactttgtg ggctcgagct 1080 aagtccgttg atctgagtag caaaaccgaa ataacttcat gttatatgtt catatggaag 1140 ctcttttatg cagagtactt gctgttacct tttttgaagt ga 1182 <210> 2 <211> 1467 <212> DNA <213> Arabidopsis thaliana <400> 2 atggagatac ggagcttgat tgtttctatg aaccctaatt tatcttcctt tgagctctct 60 cgccctgtat ctcctctcac tcgctcacta gttccgttcc gatcgactaa actagttccc 120 cgctccattt ctagggtttc ggcgtcgatc tccaccccga atagtgaaac tgacaagatc 180 tccgttaaac ctgtttacgt cccgacgtct cccaatcgcg aactccggac tcctcacagt 240 ggataccatt tcgatggaac acctcggaag ttcttcgagg gatggtattt cagggtttcc 300 atcccagaga agagggagag tttttgtttt atgtattctg tggagaatcc tgcatttcgg 360 cagagtttgt caccattgga agtggctcta tatggaccta gattcactgg tgttggagct 420 cagattcttg gcgctaatga taaatattta tgccaatacg aacaagactc tcacaatttc 480 tggggagatc gacatgagct agttttgggg aatactttta gtgctgtgcc aggcgcaaag 540 gctccaaaca aggaggttcc accagaggaa tttaacagaa gagtgtccga agggttccaa 600 gctactccat tttggcatca aggtcacatt tgcgatgatg gccgtactga ctatgcggaa 660 actgtgaaat ctgctcgttg ggagtatagt actcgtcccg tttacggttg gggtgatgtt 720 ggggccaaac agaagtcaac tgcaggctgg cctgcagctt ttcctgtatt tgagcctcat 780 tggcagatat gcatggcagg aggcctttcc acagggtgga tagaatgggg cggtgaaagg 840 tttgagtttc gggatgcacc ttcttattca gagaagaatt ggggtggagg cttcccaaga 900 aaatggtttt gggtccagtg taatgtcttt gaaggggcaa ctggagaagt tgctttaacc 960 gcaggtggcg ggttgaggca attgcctgga ttgactgaga cctatgaaaa tgctgcactg 1020 gtttgtgtac actatgatgg aaaaatgtac gagtttgttc cttggaatgg tgttgttaga 1080 tgggaaatgt ctccctgggg ttattggtat ataactgcag agaacgaaaa ccatgtggtg 1140 gaactagagg caagaacaaa tgaagcgggt acacctctgc gtgctcctac cacagaagtt 1200 gggctagcta cggcttgcag agatagttgt tacggtgaat tgaagttgca gatatgggaa 1260 cggctatatg atggaagtaa aggcaaggtg atattagaga caaagagctc aatggcagca 1320 gtggagatag gaggaggacc gtggtttggg acatggaaag gagatacgag caacacgccc 1380 gagctactaa aacaggctct tcaggtccca ttggatcttg aaagcgcctt aggtttggtc 1440 cctttcttca agccaccggg tctgtaa 1467 <210> 3 <211> 393 <212> PRT <213> Arabidopsis thaliana <400> 3 Met Glu Ser Leu Leu Ser Ser Ser Ser Leu Val Ser Ala Ala Gly Gly   1 5 10 15 Phe Cys Trp Lys Lys Gln Asn Leu Lys Leu His Ser Leu Ser Glu Ile              20 25 30 Arg Val Leu Arg Cys Asp Ser Ser Lys Val Val Ala Lys Pro Lys Phe          35 40 45 Arg Asn Asn Leu Val Arg Pro Asp Gly Gln Gly Ser Ser Leu Leu Leu      50 55 60 Tyr Pro Lys His Lys Ser Arg Phe Arg Val Asn Ala Thr Ala Gly Gln  65 70 75 80 Pro Glu Ala Phe Asp Ser Asn Ser Lys Gln Lys Ser Phe Arg Asp Ser                  85 90 95 Leu Asp Ala Phe Tyr Arg Phe Ser Arg Pro His Thr Val Ile Gly Thr             100 105 110 Val Leu Ser Ile Leu Ser Val Ser Phe Leu Ala Val Glu Lys Val Ser         115 120 125 Asp Ile Ser Pro Leu Leu Phe Thr Gly Ile Leu Glu Ala Val Val Ala     130 135 140 Ala Leu Met Met Asn Ile Tyr Ile Val Gly Leu Asn Gln Leu Ser Asp 145 150 155 160 Val Glu Ile Asp Lys Val Asn Lys Pro Tyr Leu Pro Leu Ala Ser Gly                 165 170 175 Glu Tyr Ser Val Asn Thr Gly Ile Ala Ile Val Ala Ser Phe Ser Ile             180 185 190 Met Ser Phe Trp Leu Gly Trp Ile Val Gly Ser Trp Pro Leu Phe Trp         195 200 205 Ala Leu Phe Val Ser Phe Met Leu Gly Thr Ala Tyr Ser Ile Asn Leu     210 215 220 Pro Leu Leu Arg Trp Lys Arg Phe Ala Leu Val Ala Ala Met Cys Ile 225 230 235 240 Leu Ala Val Arg Ala Ile Ile Val Gln Ile Ala Phe Tyr Leu His Ile                 245 250 255 Gln Thr His Val Phe Gly Arg Pro Ile Leu Phe Thr Arg Pro Leu Ile             260 265 270 Phe Ala Thr Ala Phe Met Ser Phe Phe Ser Val Val Ile Ala Leu Phe         275 280 285 Lys Asp Ile Pro Asp Ile Glu Gly Asp Lys Ile Phe Gly Ile Arg Ser     290 295 300 Phe Ser Val Thr Leu Gly Gln Lys Arg Val Phe Trp Thr Cys Val Thr 305 310 315 320 Leu Leu Gln Met Ala Tyr Ala Val Ala Ile Leu Val Gly Ala Thr Ser                 325 330 335 Pro Phe Ile Trp Ser Lys Val Ile Ser Val Val Gly His Val Ile Leu             340 345 350 Ala Thr Thr Leu Trp Ala Arg Ala Lys Ser Val Asp Leu Ser Ser Lys         355 360 365 Thr Glu Ile Thr Ser Cys Tyr Met Phe Ile Trp Lys Leu Phe Tyr Ala     370 375 380 Glu Tyr Leu Leu Leu Pro Phe Leu Lys 385 390 <210> 4 <211> 488 <212> PRT <213> Arabidopsis thaliana <400> 4 Met Glu Ile Arg Ser Leu Ile Val Ser Met Asn Pro Asn Leu Ser Ser   1 5 10 15 Phe Glu Leu Ser Arg Pro Val Ser Pro Leu Thr Arg Ser Leu Val Pro              20 25 30 Phe Arg Ser Thr Lys Leu Val Pro Arg Ser Ile Ser Arg Val Ser Ala          35 40 45 Ser Ile Ser Thr Pro Asn Ser Glu Thr Asp Lys Ile Ser Val Lys Pro      50 55 60 Val Tyr Val Pro Thr Ser Pro Asn Arg Glu Leu Arg Thr Pro His Ser  65 70 75 80 Gly Tyr His Phe Asp Gly Thr Pro Arg Lys Phe Phe Glu Gly Trp Tyr                  85 90 95 Phe Arg Val Ser Ile Pro Glu Lys Arg Glu Ser Phe Cys Phe Met Tyr             100 105 110 Ser Val Glu Asn Pro Ala Phe Arg Gln Ser Leu Ser Pro Leu Glu Val         115 120 125 Ala Leu Tyr Gly Pro Arg Phe Thr Gly Val Gly Ala Gln Ile Leu Gly     130 135 140 Ala Asn Asp Lys Tyr Leu Cys Gln Tyr Glu Gln Asp Ser His Asn Phe 145 150 155 160 Trp Gly Asp Arg His Glu Leu Val Leu Gly Asn Thr Phe Ser Ala Val                 165 170 175 Pro Gly Ala Lys Ala Pro Asn Lys Glu Val Pro Pro Glu Glu Phe Asn             180 185 190 Arg Arg Val Ser Glu Gly Phe Gln Ala Thr Pro Phe Trp His Gln Gly         195 200 205 His Ile Cys Asp Asp Gly Arg Thr Asp Tyr Ala Glu Thr Val Lys Ser     210 215 220 Ala Arg Trp Glu Tyr Ser Thr Arg Pro Val Tyr Gly Trp Gly Asp Val 225 230 235 240 Gly Ala Lys Gln Lys Ser Thr Ala Gly Trp Pro Ala Ala Phe Pro Val                 245 250 255 Phe Glu Pro His Trp Gln Ile Cys Met Ala Gly Gly Leu Ser Thr Gly             260 265 270 Trp Ile Glu Trp Gly Gly Glu Arg Phe Glu Phe Arg Asp Ala Pro Ser         275 280 285 Tyr Ser Glu Lys Asn Trp Gly Gly Gly Phe Pro Arg Lys Trp Phe Trp     290 295 300 Val Gln Cys Asn Val Phe Glu Gly Ala Thr Gly Glu Val Ala Leu Thr 305 310 315 320 Ala Gly Gly Gly Leu Arg Gln Leu Pro Gly Leu Thr Glu Thr Tyr Glu                 325 330 335 Asn Ala Ala Leu Val Cys Val His Tyr Asp Gly Lys Met Tyr Glu Phe             340 345 350 Val Pro Trp Asn Gly Val Val Arg Trp Glu Met Ser Pro Trp Gly Tyr         355 360 365 Trp Tyr Ile Thr Ala Glu Asn Glu Asn His Val Val Glu Leu Glu Ala     370 375 380 Arg Thr Asn Glu Ala Gly Thr Pro Leu Arg Ala Pro Thr Thr Glu Val 385 390 395 400 Gly Leu Ala Thr Ala Cys Arg Asp Ser Cys Tyr Gly Glu Leu Lys Leu                 405 410 415 Gln Ile Trp Glu Arg Leu Tyr Asp Gly Ser Lys Gly Lys Val Ile Leu             420 425 430 Glu Thr Lys Ser Ser Met Ala Ala Val Glu Ile Gly Gly Gly Pro Trp         435 440 445 Phe Gly Thr Trp Lys Gly Asp Thr Ser Asn Thr Pro Glu Leu Leu Lys     450 455 460 Gln Ala Leu Gln Val Pro Leu Asp Leu Glu Ser Ala Leu Gly Leu Val 465 470 475 480 Pro Phe Phe Lys Pro Pro Gly Leu                 485 <210> 5 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tcacttcaaa aaaggtaaca gcaagta 27 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 catgccatgg agtctctgct ctctagttct 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gggtcacctc acttcaaaaa aggtaacagc 30 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttacagaccc ggtggcttga agaaagg 27 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 catgccatgg agatacggag cttgattgtt 30 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gggtcacctt acagacccgg tggcttgaa 29 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gtttctgata tatctccttt a 21 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 catgcttaac gtaattcaac 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 catgccatgg agatacggag cttgattgtt 30 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gaacccttcg gacactcttc tgtta 25  

Claims (2)

A gamma tocopherol content was transformed with a recombinant vector comprising a gene encoding a TC / VTE1 (tocopherol cyclase) protein derived from Arabidopsis thaliana involved in the synthesis of tocopherol, consisting of the amino acid sequence represented by SEQ ID NO: 4. Transgenic lettuce characterized by an increased content. A method of increasing gamma tocopherol content by overexpressing in a lettuce a gene encoding a TC / VTE1 protein from Arabidopsis thaliana involved in the synthesis of tocopherol, consisting of the amino acid sequence represented by SEQ ID NO: 4.

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