KR100813118B1 - Promoter for skin preferential expression in fruit - Google Patents

Abstract

A promoter is provided to show skin preferential expression characteristic on fruit, thereby being useful for controlling the characteristics of the fruit such as color of the skin and tissue. A promoter with skin preferential expression characteristic in fruit consists of a nucleotide sequence described in SEQ ID : NO. 1. A vector comprises the promoter and a foreign gene operatively linked to the promoter. A transgenic plant is transformed by the vector. A method for transforming a plant comprises a step of expressing the foreign gene in the plant by transforming the plant using the vector.

Description

Promoter for Skin Preferential Expression in Fruit

1A-1C show the nucleotide sequence of the MdANS1 gene and the structure of MdANS1 :: GUS .

1A shows the restriction map of the MdANS1 genome clone. The 1.5 kb Eco RI fragment, denoted pANS1.5, contains the ATG start codon with the upstream portion and pANS2.0 contains the remaining downstream portion. The MdANS1 coding part is marked with a thick black box and the intron is marked with a white box. E, Eco RI; B, BamH I; S, Sac I. The two BamH I sites ^a are located close to each other. Another Sac I site ^b is located upstream near Eco RI.

1B shows the nucleotide sequence and the inferred amino acid sequence of the MdANS1 gene. Non-coding regions are shown in lowercase and coding regions are shown in uppercase. The putative amino acid sequence of the MdANS1 gene is shown below the nucleotide sequence. The numbers on the left and right side indicate the positions of the nucleotides and amino acids, respectively. The first nucleotide of MdANS1 cDNA is shown in bold and designated as +1. Presumed CAAT and TATA boxes of promoter sites, and polyadenylation signals are underlined.

1C shows chimeric gene constructs designed to express GUS under MdANS1 in transgenic tobacco. This gene cassette was inserted into the binary vector pBI101.2 and introduced into tobacco leaf discs by Agrobacterium -mediated transformation. RB is the right border of T-DNA; P _NOS is the nopaline synthase promoter; npt II is the neomycin phosphotransferase gene; T _NOS is a nopaline synthase terminator; LB is the left boundary of T-DNA.

Figure 2 is a photograph showing MdANS1 promoter-induced GUS expression in transformed tobacco plants. Blue precipitates indicate the location of GUS activity. Panel A of FIG. 2 is a young bud. Magnification shows that there is activity in the trichome of the sepal. Bar = 1 mm. Panel B in Fig. 2 shows flowers before flowering. The magnification shows that activity is seen in vascular tissues as well as the dendritic structure of the petal. Bar = 2 mm. Panel C of FIG. 2 shows a cross section of the fruit in development. Magnification shows that there is activity in the seed coat. Bar = 1 mm. Panel D in Figure 2 is the lower part of the fruit in the developmental stage. Old calyx pieces were removed from the fruit for a picture of the GUS-expression. Bar = 1 mm. P is a petal; R is the jaw; S is calyx; Se is a seed in the developmental stage.

The present invention relates to a promoter having epidermal tissue specific expression characteristics of the fruit of the plant.

The development of color in flowers and fruits is an important property in the fitness of plants. Coloration results from the mixing of chlorophyll, carotenoids, and flavonoids. Anthocyanins are secondary products belonging to a subclass of flavonoids. They function as pigments in flowers and fruits to entice insects for pollination or to help seed spread (Holton and Cornish, 1995; Springob et al, 2003).

Anthocyanins and other flavonoids act as protective agents against UV light and pathogens or as signaling molecules in the interaction between bacteria and plants (Harbone and Grayer, 1994; Holton and Cornish, 1995; Dixon and Steele, 1999; Springob et al., 2003; Kim et al., 2006). In addition to anthocyanins, other pigments such as flavonols and carotenoids, metal complexes, pH of vacuole, and the shape of cells can affect the phenotype of the resulting pigmentation (Forkmann, 1994; Holton and Cornish). , 1999).

Anthocyanin biosynthetic pathways are known for secondary metabolites (Dixon and Steele, 1999).

This is called Petunia hybrida ) and Snapdragon ( Antirrhinum) majus flowers and corn ( Zea) mays ) has been extensively studied in grains (Holton and Cornish, 1995). Among the genes and enzymes studied in this biosynthetic pathway, anthocyanidin synthase (ANS) converts leucoanthocyanidins to anthocyanidins, the first pigment compound in the anthocyanin synthesis pathway. Very important (Springob et al., 2003).

ANS transcripts from forsythia (Forsythia) have only been found in the calyx pieces (sepals) was not found in the anthers (anthers) or petals (petal) (Rosati et al. , 1999). In etiolated plants of Arabidopsis , the ANS gene was induced to the highest level by white light after the flavonol synthase gene (FLS), which indicates that the ANS gene is a late gene in the biosynthetic pathway. Imply (Pelletier et al., 1997). Perilla leaf detected by the red of the red Forma (forma) (Perilla) ANS The expression of the gene was comparably induced in forma by high intensity white light (Gong et al., 1997).

Similarly, transcripts of the apple ANS gene were selectively detected in the epidermal tissue of red fruits rather than blue, which is regulated by light (Kim et al., 2003).

Regulatory genes in the anthocyanin biosynthetic pathway have also been identified (Springob et al., 2003). Among these genes, well-characterized genes are those encoding MYB and basic helix-loop-helix (bHLH) that influence the intensity and pattern of expression (Holton and Cornish, 1995; Springob et al., 2003 ). For example, the Forsythia ANS gene contains several cis-elements involved in the activation and photoreaction of the anthocyanin gene (Rosati et al., 1999). In maize, the C1-binding site with the second contiguous position adjacent functions in the activation of the ANS gene (Lesnick and Chandler, 1998).

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors tried to find a promoter having a function of regulating gene expression selectively in the epidermal tissue of fruit, and cloned MdANS1 , anthocyanidin synthase ( ANS ) gene of apple , which gene Promoter region and GUS GUS in the reproductive system of tobacco plants transformed with MdANS1 :: GUS By confirming the activity, the present invention was completed.

Accordingly, it is an object of the present invention to provide a promoter having the property of specifically expressing the epidermal tissue of a fruit of a plant.

Another object of the present invention is to provide a vector comprising the promoter of the present invention and the desired foreign gene.

Another object of the present invention to provide a transformed plant transformed by the vector of the present invention.

Still another object of the present invention is to provide a method for transforming a plant by the vector of the present invention.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, there is provided a promoter consisting of the nucleotide sequence set forth in SEQ ID NO: 1 having the property of specifically expressing a gene in fruit epidermal tissue of a plant.

The present inventors tried to find a promoter having a function of regulating the gene to specifically express the epidermal tissue of the fruit of the plant, and cloned MdANS1 , an anthocyanidin synthase ( ANS ) gene of apple , GUS in the reproductive organs of transgenic tobacco plants with the MdANS1 :: GUS fusion gene promoter and the GUS gene The activity was confirmed.

The term "specifically" as used herein means that the expression activity of the promoter is higher in a particular tissue than at least one or more other tissues in the same plant. The level of expression activity of a promoter is assessed by comparing the expression level of the promoter in tissues previously measured using those methods commonly used to those in other tissues. In general, the expression level of a promoter is measured by the amount of gene product expressed under the control of the promoter, for example, protein and RNA.

The term "promoter" as used herein refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoter sites are readily recognized by those skilled in the art. That is, an estimated start codon containing an ATG motif has been identified, and an upstream from the start codon is an estimated promoter site. The promoter consists of upstream and downstream elements. Adjacent elements typically include a TATA box that allows RNA polymerase II to initiate the synthesis of RNA at a suitable transcription initiation site. Far-field elements include regulatory sequences, also called enhancers, which are additional regulatory elements involved in tissue specific or time specific expression upstream of the TATA box. Enhancers are DNA sequences capable of promoting the activity of a promoter, and may be elements of the promoter or elements of heterologous that are inserted to enhance the tissue-specificity or expression level of the promoter. The promoter may be derived entirely from the original gene, or may be composed of different elements from different promoters found in nature, and may even include synthetic DNA. Those skilled in the art will appreciate that each of the different promoters can direct the expression of the gene in different tissues or cell types, at different stages of development, or in response to different environmental conditions. Promoters that express genes most of the time in most cell types are called "constitutive promoters." New types of new promoters that are useful in plant cells are constantly being found; A number of promoter examples are disclosed in compilations by Okamuro and Goldberg (1989, Biochemistry of Plants 15: 1-82). In most cases, it is recognized that certain fragments of DNA fragments have the same promoter activity because the exact boundaries of the regulatory sequences are not fully defined.

According to another aspect of the present invention, there is provided a vector comprising a promoter consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a foreign gene operatively linked with the promoter.

As used herein, the term “operably linked” means a functional binding between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an arrangement of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and / or translation of said other nucleic acid sequence.

The term "desired foreign gene" herein is not limited to a particular gene, and may be selected according to the desired application or phenotype to be achieved. In the present invention, it is preferable to select a gene useful for improving the properties of the surface texture of the fruit, for example, the color or surface texture of the fruit.

Vectors in the present invention can be constructed through a variety of methods known in the art, specific methods thereof are described in Sambrook et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage λ (pLλ) Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the genome of the mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

Vectors of the present invention may include antibiotic resistance genes commonly used in the art as optional markers, for example ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be used any conventionally used in the art for the gene introduction of plants, for example, the ubiquitin promoter of corn, cauliflower mosaic virus (CaMV) 35S promoter, nopalin synthase (nos) promoter, pigwart mosaic virus 35S promoter, sugacrein bacilli foam virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis- Photoinducible promoter of phosphate carboxylase small subunit (ssRUBISCO), rice cytosolic triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of arabidopsis and octopine synthase promoter It includes.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research, 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion, or CaMV 35S terminator.

Optionally, the vectors of the present invention additionally carry genes encoding reporter molecules (eg, luciferase and β-glucuronidase). In addition, the vectors of the present invention may be selected as antibiotic markers (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.). Gromycin phosphotransferase (hpt), and the like).

According to another aspect of the present invention, there is provided a transformed plant transformed with the vector.

According to another aspect of the present invention, there is provided a method for transforming a plant comprising the step of transforming the plant with the vector to express the foreign gene in the plant.

Host cells capable of stable and continuous cloning and expression of the vectors of the present invention are known in the art and can be used with any host cell, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

In the case of transforming a vector of the present invention into eukaryotic cells, as a host cell, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. On the other hand, since the vector of the present invention is highly useful in plants, the transformants include not only cells but also callus derived from plant cells or tissues.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, fine injection method (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973)), Electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene, 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 (1985)), and gene balm (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) Can be injected into the host cell.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982) ), Particle bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011) Include. Of these, Agrobacterium-mediated transformation is most preferred.

It is known how to transform a dicotyledonous plant using Agrobacterium trimmers and obtain a transformed plant, and a method of obtaining a transformed plant for various other plants is known. See US Pat. Nos. 5,004,863 and 5,159135; See US Pat. Nos. 5,569,834 and 5,416011 for soy; Brassica is described in US Pat. No. 5,463,174; For peanuts, see Cheng et al. (1996) Plant Cell Rep . 15: 653-657, McKently et al. (1995) Plant Cell Rep . 14: 699-703; For papaya, see Ling, K. et al. (1991) Bio / technology 9: 752-758); and pea (Grant et al. (1995) Plant Cell Rep . 15: 254-258.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that are transformed and stably contain marker genes that confer selective resistance are grown and divided in the cultures described above. Exemplary labels include, but are not limited to, the hygromycin phosphotransferase gene, glycophosphate resistance gene, and neomycin phosphotransferase (nptII) systems.

Methods of developing or regenerating plants from plant protoplasts or various implants are well known in the art. Development or regeneration of plants comprising foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens-binary vector system.

Transformation of plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker, A. etal., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983)). . More preferably, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986) An et al., 1988 and Lee et al., 1999).

Binary vectors suitable for the present invention include (i) a promoter operating in a plant; (Ii) structural genes operably linked to said promoter; And (iii) a polyadenylation signal sequence. Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and glucuronidase). Examples of promoters used in binary vectors include, but are not limited to, CaMV 35S promoter, 1 promoter, 2 promoter and nopalin synthase (nos) promoter.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture with an implant impregnated with a culture of Agrobacterium trimmers. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed by Agrobacterium trimerization are re-differentiated in regeneration medium, which finally forms transgenic plants.

Plants transformed according to the present invention is confirmed whether or not transformed by methods known in the art. For example, PCR can be performed using DNA samples obtained from tissues of transformed plants to identify foreign genes inserted into the genome of the transformed plants. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention provides a promoter for specifically expressing genes in the epidermal tissue of fruits.

(Ii) The promoter of the present invention can be usefully used to control characteristics such as color in the epidermal tissue of fruits.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experiment method

Bacterial strains

E. coli strains MC1000 and XL-1 Blue MRF 'were used as host strains for molecular cloning. The f1 helper phage R408 was used to excision pBluescript phagemid from the λUNI-Zap XR vector (Stratagene, USA). E. coli strain XL1-Blue MRA (P2) was used as a host for lambda phage preparation after screening the genome library.

Screening of the Genome Library

The apple genome library was obtained from Dr. Woo-Taek Kim of Yonsei University in Korea. Libraries were prepared from young apple leaves using Bam HI-cleaved λDASHII vector (Stratagene) according to the manufacturer's instructions. Phage of the library was screened by plaque hybridization with a MdANS1 probe (Accession Number AF117269) labeled with [α- ³² p] dCTP. After the third screen, positive plaques were isolated and restriction enzyme fragments of the recombinant clones were subcloned into the pBluescript SK (-) vector (Stratagene).

DNA  Sequence analysis

DNA sequencing was performed according to the dioxyoxynucleotide chain termination method using a Thermo Sequenase Cyle Sequencing Kit (Amersham, UK) and an Autosequencer (ABI, USA). The sequences were compared to those of the database using the BLAST program (Altschul et al., 1990) and DNASIS and PROSIS software (Hitachi, Japan). The putative DNA-protein binding site in the promoter sequence was investigated using the Mat-Inspector program and standard settings (Standard settings, Core similarity, 0.80; matrix similarity, 0.85).

ANS  Analysis of genome clones and ANS :: GUS Manufacture

The genome clone corresponding to MdANS1 cDNA was isolated and a 1.5 kb Eco RI fragment comprising the MdANS1 5′-side was subcloned into pBluescript SK (−) to prepare pANS1.5. pSK126 was prepared by translating the Sal I / Xba I fragment of pANS1.5 to pBI101.2 (Accession number U12668; Jefferson et al.,). Then, a vector a freeze-Agrobacterium tyumeo Patience by thawing method (freeze-thaw method) (Agrobacterium tumefaciens ) was introduced into strain LBA4404.

Tobacco transformation and GUS  analysis

Nicotiana tabacum var. SR1 was transformed with Agrobacterium tumefaciens by leaf disk transformation. Flowers, buds and leaves of transformed tobacco plants grown in greenhouses were used for GUS analysis according to the method described by Jefferson et al. (1987). Tissues were 0.1% (w / v) 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Glu), 10 mM EDTA, 0.5% (v / v) Was incubated in the dark at 37 ° C. for 10-13 hours in 100 mM sodium phosphate buffer (pH 7.0) containing Triton X-100, 5 mM potassium ferrocyanide, and 20% methanol. After staining the tissues, they were washed with ethanol at 50 ° C. for 5 hours and observed under a microscope under dark conditions.

Experiment result

Apple ANS  Genome clone isolation

The ANS gene ( MdANS1 ), which is selectively expressed in the epidermal tissue of Fuji, a red apple variety, has already been studied (Kim et al., 2003). To better understand the regulatory mechanism for this tissue specific expression, genome clones were isolated. About 3 × 10 ⁵ plaques of the genome library were screened by plaque hybridization using MdANS1 cDNA as a probe. After the third screening, nine recombinant plaques were obtained and divided into four groups based on their restriction maps (data not shown). It was found that λANS1 , a genome clone approximately 12 kbps long, corresponds to MdANS1 cDNA. The 1.5-kbp Eco RI subclone of λANS1 (pANS1.5) included a 5 ′ side sequence site with a portion of the coding site (FIG. 1A).

The adjacent 2.0-kbp Eco RI subclone, pANS2.0, included the remainder along with the 194 bp intron and 3 'side site. These genome structures, including introns, are similar to those found in Arabidopsis, Forsythia and Petunia, but differ from those found in corn (Furtek et al., 1998; Menssen et al., 1990; Pelletier et al., 1997; Rosati et al., 1999) The introns in the present invention contained consensus GT and AG sequences at the 5 'and 3' ends, respectively.

ANS  Of promoter sign Silico ( In Silico )  analysis

Putative CAAT box sequence CAAT, and TATA box sequence TATAAAA is MdANS1 It was found in the 5 'side sequence of MdANS1 at -112 / -109 and -91 / -85 from the cDNA 5' end, respectively. The Myb-class P activator binding site, CCAACC (positions -847 / -842) and two similar sequences, as well as several cis-elements important for photoreaction or flavonoid biosynthesis-specific expression were located (Table 1). . In corn husks, P protein activates the transcription of the A1 gene required for 3-deoxyflavonoid biosynthesis (Pelletier and Shirley, 1996). The genes we found also included cis-elements (Martin et al., 1991; Grotewold et al., 1994) required for C1 and MYB.Ph3 binding at three positions (Table 1). Cis-elements for C1, P and MYB.Ph3 binding were also found in the Forsythia ANS promoter (Rosati et al., 1999). In addition, in the MdANS1 promoter of the present invention (Table 1), G-box (CACGTG) and GT-1 box (GGTTAA) found in promoters primarily controlled by light (Solano et al., 1995; Terzaghi and Other elements, such as Cashmore, 1995). Normally GT-1 sites are found in front and back side by side, and the spacing between these sites is very important (Terzaghi and Cashmore, 1995). In the MdANS1 promoter, the GT-1 site at the -1042 position is 22-b away from the SBF-1 binding site, a sequence closely related to the GT-1 binding site. The promoter region contains three G box core sequences that overlap with the ABA-reactive element (ABRE). It was confirmed that MdANS1 is photo-inducible (Kim et al. 2003). Transcription factors that bind to putative cis-elements may mediate activation or inhibition of transcription of the MdANS1 gene.

TABLE 1

Putative cis-elements observed in the MdANS1 promoter of the present invention

Putative cis-elements observed at 1398 bp upstream from the 5 'end of MdANS1 cDNA.

the ^b nucleotide sequence represents the core consensus sequence reported in promoters from other plant species.

^c GT-1 boxes and ^d ABRE core sequences are underlined.

the ^e sequence is the sequence found in the reverse direction compared to the direction of the MdANS1 open reading frame.

Apples in Transgenic Tobacco Plants ANS :: GUS Expression of

Transformed plants were prepared to investigate the role of the MdANS1 promoter in tissue-specific expression. Since apples are difficult to transform, the tobacco plant system was used. The ANS promoter site was translated to the GUS gene of the binary vector pBI101.2 (FIG. 1C). Based on the PCR assay of the ANS :: GUS structure, 6 out of 10 regenerated plants were identified as transformants (data not shown). Expression of these fused genes was analyzed by histochemical GUS assay. There was little GUS activity in the vegetative growth phase of the transformed tobacco plants, which is consistent with the results from the Northern analysis of MdANS1 . In contrast, in the reproductive phase, GUS activity is expressed in the reproductive organs: the sepal of the floral bud, the petals of mature flowers, the receptacle and various tissues from the seed of the developmental stage. Was detected in (FIG. 2). In the calyx fragment, GUS activity was found mainly in the trichome and weakly in the epithelial tissue. Similarly, in mature petals, activity was greater in dendritic structure than in epithelial tissue. The petals also showed strong activity in the vascular tissue. In the fruit of the developmental stage, the activity of immature seeds in blunt serrated epithelial cells was remarkable, but weak in placenta. Strong activity was observed in the middle of the stem. The high activity in the reproductive organs and the low activity in the nutrient organs of the transformed tobacco is a result consistent with the reproductive organ selective expression of MdANS1 (Kim et al. 2003) previously studied.

GUS activity was very high in the immature seeds of the transgenic tobacco of the present application (FIG. 2), which may be associated with anthocyanin production in the seed coat in the developmental stage. Phaseolus In vulgaris , the CHS15 promoter-derived GUS gene exhibits GUS expression in epithelial cells of seeds at developmental stages from transformed tobacco plants (Lawton et al., 1991).

On the other hand, the present invention was able to detect only the weak activity in the fruit epidermis of the transformed tobacco, perhaps because tobacco and apple is different in the fruit development process. In other words, tobacco fruit is a dehiscent capsule that develops from the ovary, while apple fruit is an indehiscent flesh, a hypthium that develops into fruit milk tissue (Fahn, 1990).

As described in detail above, the present invention provides a promoter having a characteristic of specifically expressing a gene in the epidermal tissue of a fruit. The promoter of the present invention can be usefully used to control the characteristics of the color, tissue, etc. of the epidermal tissue of the fruit.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (4)

A promoter of fruit epidermal tissue specific expression properties consisting of the nucleotide sequence set forth in SEQ ID NO: 1. A vector comprising (a) the promoter of claim 1 and (b) a foreign gene operatively linked to the promoter. Transgenic plant transformed by the vector of claim 2. A method for transforming a plant, comprising the step of transforming the plant with the vector of claim 2 to express the foreign gene in the plant.

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