KR101369526B1 - Promoter for directing embryo and seed inner integument specific expression - Google Patents

Abstract

The present invention provides a promoter of the Arabidopsis At5g54000 gene (SEQ ID NO: 1), which induces embryo tissue and endothelial specific expression of plant seeds, a plant embryo tissue and seed endothelial specific expression vector comprising the promoter and The present invention relates to a method for producing a target protein by expressing a foreign gene in the embryo tissue and endothelial of plant seeds using a vector. The embryo tissue and seed endothelial specific expression promoter according to the present invention specifically induces expression only in embryo tissue and seed endothelial inside the dicot plant seed. Thus, this promoter can induce gene expression in the embryonic tissue and seed endothelium of mature seeds of dicotyledonous plants, improve the development of transformed plants to produce useful medical and industrial proteins in the same tissue, and improve seed conservation and germination. It can be usefully used for the development of transformed plants.

Description

Embryonic tissue and seed inner integument specific promoter

The present invention relates to embryo tissue and seed inner integument specific promoters. In more detail, the present invention is a base sequence that induces seed endothelial tissue and seed endothelial specific expression of a dicotyledonous plant of the base sequence which is a promoter of the Arabidopsis At5g54000 gene, plant embryo tissue and seed endothelial expression recombinant including the base sequence The present invention relates to a vector and a method for producing a target protein by expressing a foreign gene in embryonic tissue and seed endothelium of a dicotyledonous plant seed using the vector.

Crop molecular breeding can use all kinds of genes as a material and it is possible to control the effect of breeding infinitely by dragging the breeding technique which was possible only by the existing genome unit as a gene unit, Technology. The growing area and cultivation of GM crops using these crop molecular breeding technologies has been continuously increasing worldwide, and the global market size of commercialized GM varieties has grown from US $ 4 billion in 2002 to US $ 10 billion in 2010. It is 15% of the total global seed market size of $ 30 billion, including soybeans, corn, cotton, and rapeseed. In order to maximize the effect of genetically modified crops, the expression of exogenously inserted genes must be finely regulated by the developmental stage and tissue of the plant, and various promoters should be developed for this purpose.

Therefore, since the early 1980s, studies on promoters that regulate expression of plant genes have been conducted. The promoter of the cauliflower mosaic virus (Cauliflower mosaic virus) would induce a strong expression in plant tissue before possibly being presented (Hohn et al., 1982, Curr. Topics Microbiol . Immunol . 96: 193-236), the nucleotide sequence of the promoter was revealed (Odell et al., 1985, Nature 313: 810-812), and strong expression was demonstrated in plants (Sanders et al., 1987, Nucleic) Acids Res . 15: 1543-58). The CaMV 35S (patent no .: JP1993192172-A1) promoter has since become the most popular universal promoter in plants.

On the other hand, studies on the expression of tissue-specific promoters that induce the expression of plant-specific tissues have been steadily progressed, so that root-tissue specific promoters (Elmayan and Tepfer, 1995, Transgenic Research 4: 388-396; Xu et al., 1995, Plant Molecular Biology , 27: 237-248; Nitz et al., 2001, Plant Sci . 161: 337-346; Vaughan et al., 2006, Journal of Experimental Botany 57: 3901-3910; Vijaybhaskar et al., 2008, Journal of Biosciences , 33: 185-193; Jeong et al., 2010, Plant Physiology , 153: 185-197; Noh et al., 2012, Transgenic Research 21: 265-278), storage root specific promoter (Herma et al., 2012, Planta, 236: 1955-1965; Noh et al., 2012, Transgenic Research 21: 265-278), flower tissue specific promoter (van der Meer et al., 1990, Plant Mol . Biol . 15: 95-109; Ruiz-Rivero and Prat, 1998, Plant Mol . Biol . 36: 639-648; Chiou and Yeh, 2008, Plant Molecular Biology , 66: 379-388; Verdonk et al., 2008, Plant Biotechnology Journal 6: 694-701; Liu et al., 2011, Plant Cell Reports 30: 2187-2194), anther specific promoter (van Tunen et al., 1990, Plant Cell 2: 393-401; Kato et al., 2010, Plant Molecular Biology Reports 28: 381-387), seed specific promoters (Bustos et al., 1989, Plant Cell 1: 839-853; Kridl et al., 1991, Seed Sci Res , 1: 209-219; Washida et al., 1999, Welham and Domoney, 2000, Plant Sci . 159: 289-299; Plant Mol . Biol . 40: 1-12; Furtado et al., 2008, Plant Biotechnology Journal 6: 679-693; Chung et al., 2008, Plant Cell Reports , 27: 29-37).

In recent years, specific expression of plant tissue has been further subdivided in order to more finely control the expression of the inserted gene in the production of genetically modified crops. For seed-specific promoters, seed-specific promoters have been developed. Embryo (Dishi et al., 2006, In Vitro Cellular and Developmental Biology , 42: 432-438; Kim et al., 2011, B iochemical and Biophysical Research Communications , 408: 78-83; Jose-Extanyol and Puigdomenech, 2012, Plant Molecular Biology, 80:. 325-335), endosperm (endosperm) (Hu et al, 2011, Biotechnol Lett. , 33: 1465-1471; Xu et al., 2010, Plant Cell Reports , 29: 1061-1068; Vickers et al., 2006, Plant Molecular Biology , 62: 195-214; Choi et al., 2003, Plant Cell Reports , 21: 1108-1120), seed-coat (Wu et al., 2011, Plant Cell REports , 30: 75-80; Esfandiari et al., 2012, Plant Molecular Biology , DOI 10.1007 / s11103-012-9984-0) - Tissue specific promoters were developed.

Most of the promoters that induce specific expression of seed internal tissues developed to date have been developed as terminal promoters derived from terminal plant genes such as rice and wheat. However, it is already known that the original promoter activity does not appear when these terminal lobe promoters are applied to the dicots (Cornejo et al., 1993, Plant Molecular Biology , 23: 567-581; Kyozuka et al., 1993, Plant Physiology , 102: 991-1000).

Therefore, considering that the internal structure of the seeds of the dicotyledonous plants and the monocotyledonous plants is very different, and the activity of the terminal promoter is not reproduced in the dicotyledonous plants, it is possible to produce metabolic engineering and useful materials There has been a continuing need for the development of promoters that induce specific expression of seed internal tissues of dicotyledonous plants.

As a background technology of the present invention, Korean Patent No. 10-0574563 (2006.04.27) includes a plant derived from the microsomal oleic acid desaturase gene of Arabidopsis thaliana, which exhibits seed specific expression. High-efficiency expression promoters and introns and plants containing them are described for high-efficiency expression vectors and plants transformed with the expression vectors, and three chimeric genes are disclosed in Korean Patent Application Publication No. 10-1996-0702810 (1996.12.09). Ie, a first gene encoding dihydrodipicolinic acid synthase (DHDPS) that is insensitive to lysine inhibition and operably linked to a plant chloroplast transition sequence, a second gene encoding a lysine rich protein and a plant lysine protein A third gene encoding toglutarate reductase, all of which are operably linked to plant seed specific regulatory sequences Is described. In addition, Korean Patent Laid-Open Publication No. 2002-0093028 (2002.12.12) describes a Cv20oxP gene promoter that regulates the expression of genes specifically when seed generation of watermelon and a method of producing seedless plants using the same. However, there is no known promoter of the Arabidopsis-derived At5g54000 gene which induces seed internal embryonic tissue and seed endothelial specific expression of dicotyledonous plants.

Korean Patent No. 10-0574563 (April 27, 2006) Korean Patent Publication No. 10-1996-0702810 (December, 1996) Republic of Korea Patent Publication 2002-0093028 (2002.12.12)

The present invention has been made to solve the above needs, an object of the present invention is to provide a promoter of the Arabidopsis At5g54000 gene ( E mbryo and inner I ntegument, EI ) that induces seed endothelial tissue and seed endothelial specific expression of a dicotyledonous plant. To provide.

Another object of the present invention is to provide a plant transformation vector comprising a promoter of the At5g54000 gene (EI promoter) and a transformed plant transformed thereby.

It is still another object of the present invention to provide a method for producing a target protein using a plant transformed with the above expression vector.

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

According to one aspect of the present invention, the present invention provides embryo tissue and seed endothelial specific promoter of an isolated plant seed having the nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the present invention, the present invention provides an expression vector for transformation comprising the promoter.

According to another aspect of the present invention, the present invention provides E. coli comprising the expression vector for transformation.

According to another aspect of the present invention, the present invention provides a plant transformed by the expression vector.

According to another aspect of the invention, the present invention comprises the steps of: 1) transforming a plant with a recombinant expression vector comprising a promoter of the At5g54000 gene and a gene encoding a protein of interest; And 2) expressing a gene encoding a target protein in the embryo tissue and seed endothelium of the transformed plant seed to produce the target protein.

According to another aspect of the present invention, there is provided a PCR primer set forth in SEQ ID NO: 4 and SEQ ID NO: 5 for amplifying the promoter.

Hereinafter, the present invention will be described in more detail.

In order to achieve the above object, the present inventors cloned the promoter region of the At5g54000 gene showing seed specific expression using Arabidopsis TAIR / ATTED DB to induce embryonic tissue and seed endothelial specific expression of seed. The present invention was completed by confirming the activity of the same promoter in the large transformant.

Accordingly, the present invention provides embryonic tissues and seed endothelial specific promoters of isolated plant seeds having the nucleotide sequence of SEQ ID NO: 1.

In the present invention, a 'promoter' is a gene region generally located upstream of a coding region including a point at which transcription is initiated, and is commonly used for TATA boxes, CAAT box regions, and external stimuli that regulate gene expression. It is composed of an enhancer that promotes the expression of almost all genes regardless of the site, position and direction that affect gene expression in response. In addition, among these promoters, there is a promoter that induces expression in all tissues and a promoter that induces expression in a time or position-specific manner. Preferably, the promoter according to the present invention is specifically expressed in Arabidopsis of seed. At5g54000 is a gene promoter. Therefore, the promoter according to the present invention can induce the expression of the gene of interest in the embryo tissue and seed endothelial of the dicotyledonous plant seed.

The DNA sequence of the promoter is characterized in that derived from -1 to -716 base pairs from the translation start site of the coding sequence of Arabidopsis At5g54000 gene. Embryonic tissue and seed endothelial specific promoter of the present invention is a modified sequence in which not only the nucleotide sequence of SEQ ID NO: 1 but also some bases of the nucleotide sequence of SEQ ID NO: 1 are substituted, deleted or added, and the embryo tissue and seed endothelial specific promoter activity. Contains a base sequence that represents.

The present invention also provides an expression vector for transformation comprising the promoter. Preferably the present invention provides a germ tissue and seed endothelial specific recombinant expression vector of a plant prepared by operably linking a gene encoding a protein of interest downstream of the promoter.

In the present invention, an 'expression vector' refers to a plasmid, virus or other medium known in the art, into which a promoter of the present invention and a gene sequence encoding a target protein operably linked to the promoter can be inserted or introduced. it means. The gene sequence encoding the plant-specific expression promoter of the plant according to the present invention and the target protein operably linked to the promoter may be operably linked to an expression control sequence, and the operably linked gene sequence and expression control sequence May be included in an expression vector that also contains a selection marker and a replication origin.

For purposes of the present invention, "operably linked" means that the appropriate molecule is linked in such a way as to enable gene expression when bound to an expression control sequence. In addition, "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation.

The expression vector according to the present invention may be prepared by inserting an At5g54000 promoter of the present invention using a conventional vector used for protein expression and inserting a base sequence encoding a target protein downstream of the promoter. Can be. Therefore, E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5), yeast-derived plasmids (YEp13, YEp24 and YCp50) having various cloning sites as vectors that can be used in the present invention. And plasmids such as Ti plasmids, plant viral vectors, and binary vectors such as pCHF3, pPZP, pGA, and pCAMBIA families can be used. However, those skilled in the art can use any vector as long as it is a vector capable of introducing the promoter of the present invention and the base sequence encoding the target protein operably linked to the promoter into the host cell. In an embodiment of the present invention, an EI-promoter :: GUS expression vector was prepared using pBI101 vector.

In the present invention, 'target protein' refers to any target protein of external origin that is specifically expressed by the promoter of the present invention in the embryo and seed endothelial of plant seeds, preferably an exogenous protein or endogenous. Protein and reporter protein. The exogenous protein refers to a protein that does not naturally exist in a specific tissue or cell, and the endogenous protein refers to a protein expressed by a gene naturally present in a specific tissue or cell. In addition, the reporter protein is a protein expressed by the reporter gene and refers to a labeling protein that indicates its activity in the cell by its presence. In one embodiment of the present invention, the recombinant expression vector EI-promoter, in which the Arabidopsis At5g54000 promoter of the present invention is operably linked to an upstream portion of the GUS gene, to a pBI101 vector incorporating a reporter system in which a blue color gene, GUS, is linked. :: GUS was prepared. The foreign gene may be expressed by fusion with a reporter gene as necessary.

Examples of the target protein genes expressed by the transcriptional activity of the embryo and seed endothelial specific promoters of the present invention include fatty acid biosynthesis and regulatory genes when the crops to be transformed are oilseeds such as rapeseed, perilla and sesame. In the case of legumes such as soybeans and alfalfa, which have a higher production and accumulation of protein than other plants, the antibody genes can be economically added. For example, in case of corn, swine diarrheal disease, foot-and-mouth disease virus, etc. Oral vaccine (edible vaccine) genes, and for most crops that are stored and distributed as seeds for a long time, genes related to various disease resistance to increase the shelf life after harvesting, and tocopherol, beta-carotene If you want to fortify certain nutrients in the crop's seeds, And the like biosynthetic gene pathway of the quality, but is not limited to this, it may be included genes encoding various target proteins which can be adjusted by using the fold-specific promoter of the present invention.

Therefore, when the promoter of the present invention is used, in the case of a crop which consumes seeds of plants and the like, it is possible to develop a crop capable of ingesting a target protein together with seeds by expressing a useful protein of interest in a seed of a plant. Alternatively, the target protein produced in the plant may be extracted and used according to a suitable method known in the art.

The present invention also provides plant cells and plants transformed with the embryo tissue and seed endothelial specific recombinant expression vectors. A recombinant expression vector comprising a promoter of the present invention and a base sequence encoding a protein of interest linked to the promoter can be introduced into plant cells using methods known in the art. As the method for introducing the recombinant expression vector according to the present invention into a plant cell, a known method can be used without limitation, for example, calcium chloride (CaCl ₂ ) and heat shock (heat shock) method, particle total impact There are methods such as particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation by polyethylene glycol (PEG).

The present invention also provides a method for producing a transgenic plant that specifically expresses a target protein in a seed, the method comprising transforming the plant with a recombinant plant expression vector comprising a promoter of the At5g54000 gene and a gene encoding a protein of interest. To provide.

The present invention also comprises the steps of 1) transforming a plant with a recombinant expression vector comprising a promoter of the At5g54000 gene and a gene encoding a protein of interest; And 2) expressing a gene encoding a target protein in the embryo tissue and seed endothelial of the transformed plant seed to produce the target protein.

In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant. Preferably, the plant is a dicotyledonous plant. The dicotyledonous plant may be a plant such as Arabidopsis, soybean, tobacco, eggplant, pepper, potato, Rice, barley, wheat, rye, corn, sorghum, oats, and oats, which may be used in the present invention. And can be both files.

The present invention also provides a primer for PCR represented by SEQ ID NO: 4 and SEQ ID NO: 5 for cloning the Arabidopsis At5g54000 gene promoter. Cloning efficiency can be increased by using the primer.

As described above, the present invention relates to a promoter region of the Arabidopsis At5g54000 gene, which induces embryonic tissue and seed endothelial specific expression inside seeds of a dicotyledonous plant. In mature seeds of dicotyledonous plants, embryonic tissue is the largest tissue that accounts for most of the seeds. Therefore, the present invention can be effectively used for the development of transgenic plants to induce the expression of genes in embryonic tissues and seed endothelium of dicotyledonous plants or to produce useful substances in embryonic tissues.

Therefore, the promoter of the present invention is excellent in tissue specificity and time specificity, the present invention is very useful for the development of transformed plants to produce useful medical and industrial proteins, and the development of transformed plants with improved seed conservation and germination.

1 shows tissue-specific expression patterns via RT-PCR of the At5g54000 gene, which is expected to be regulated by the promoter of the present invention.
Figure 2a and 2b is a view showing a process for identifying the PCR results and the finished clone for promoter cloning for the present invention.
3 is a schematic diagram of a pBI101 vector used for promoter cloning according to the present invention.
Figure 4a is a diagram showing the results of analyzing the expression pattern of the tissue by the control of the At5g54000 gene promoter (EI promoter) according to the present invention.
Figure 4b is a diagram showing the results of analyzing the internal expression patterns of immature seed of GUS by the regulation of the At5g54000 gene promoter (EI promoter) according to the present invention. The middle drawing is the result of an enlargement of the left drawing, and the right drawing is the reference drawing used for the interpretation of the left and middle drawing.
Figure 4c is a diagram showing the results of analyzing the internal expression patterns of mature seeds of GUS by the regulation of the At5g54000 gene promoter (EI promoter) according to the present invention. The left drawing shows the seed's belly and the epidermis, and the right drawing shows the cross section of the seed.

Hereinafter, the present invention will be described in detail with reference to Examples. However, the following examples illustrate the present invention, and the scope of the present invention is not limited by the following examples.

Example  1. Selection and identification of gene (At5g54000) that specifically expresses seeds

In order to obtain a gene specifically expressed in seeds, genes selected only in seeds based on the results of affymetrix 25k (ATHI) array disclosed in TAIR / ATTED DB were selected, Genes that were unable to identify the expression pattern were selected. Among them, a gene encoding At5g54000 was selected.

RT-PCR was performed to determine whether the expression of At5g54000, which is expected to be specific for seed, is actually expressed only in seeds.

Using a TRI REAGENT ^® (TRI REAGENT ^® - RNA / DNA / PROTEIN ISOLATION REAGENT, MOLECULAR RESEARCH CENTER, INC.) To amplify the standard cDNA for RT-PCR, the 5-week Arabidopsis thaliana ecotype Columbia Extraction of total RNA (total RNA) from tissues, flowers, inflorescence, leaves, roots, immature seeds and mature seeds of 7 major Arabidopsis thaliana Respectively.

(5 μg total RNA), 2.5 μM anchored-oligo (dT) ₁₈ primer, 1 × (8 mM MgCl ₂ ) transcriptor reverse transcriptase reaction buffer, 20 U protector RNase inhibitor, 1 mM deoxynucleotide mix and 10 U transcriptor reverse transcriptase 20 μl, and then treated at 55 ° C for 70 minutes. And then treated at 85 ° C for 5 minutes to inactivate the transcriptor reverse transcriptase. A cDNA pool for each tissue was constructed as described above, and this cDNA pool was used as a template for RT-PCR.

Primers 1 and 2 (SEQ ID NOS: 2 and 3 in Table 1) were synthesized in the protein coding region based on the nucleotide sequence of Tair site and were amplified by PCR using Gene AmpPCR System 2700 (Perkin Elmer, USA) After the denaturation, the cells were subjected to 30 cycles of 95 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 30 seconds.

For RT-PCR
primer 5'-GATCAATGTAGGAGATACTATGGAGGTA-3 '
SEQ ID NO: 2 5'-TTCGACCTTGTTGATAGTAATCGA-3 '
SEQ ID NO: 3

EF1a was used as an internal control and the transcription amount of EF1a in the tissue pool was almost the same. Results of RT-PCR using the At5g54000 gene specific primer are shown in FIG. 1. As shown in FIG. 1, the At5g54000 gene was not expressed in flowers, leaves, stems, roots and immature seeds, but was strongly expressed only in mature seeds.

Example  2: Promoter of Arabidopsis At5g54000 gene Cloning

The region estimated to be the promoter of the gene was estimated to be 716 base pairs. First, genomic DNA (gDNA) of wild Arabidopsis was extracted and a primer represented by SEQ ID NO: 4 and SEQ ID NO: 5 containing Sal1 at the 5 'end and Xba1 at the 3' end was synthesized (see Table 2).

For promoter cloning
primer 5'-AAGTCGACTAACGGTAAAAAAAGTAGACC-3 '
SEQ ID NO: 4 5'-CATTCTAGATGTTTCGGAGGATCTTTG-3 '
SEQ ID NO: 5

Using the gDNA as a template, the promoter region was amplified by using primers SEQ ID NOs: 4 and 5 and PrimeSTAR ^TM HS DNA Polymerase (TAKARA BIO INC.), A DNA polymerase having proofreading function. In this case, the PCR reaction was performed by Gene Amp ^® PCR System 2700 (Perkin Elmer, USA), after initial denaturation at 95 ° C. for 5 minutes, by touch-down PCR, 30 seconds at 95 ° C., 57 ° C. In 30 seconds, 72 ℃ 1 min 20 seconds to 2 ℃ by three times, was carried out three times, 95 ℃ 30 seconds, 53 ℃ 30 seconds, 72 ℃ 1 minutes 20 seconds was carried out a total of 30 times PCR.

Confirm that the product obtained by PCR was about 732 base pairs (including restriction enzyme sequences) on agarose gel (see FIG. 2A), and the PCR product was truncated with Sal1 and Xba1 restriction enzymes, and then the pBI101 vector containing the GUS reporter gene. The vector was cloned into SalI and XbaI sites. Colony-PCR was then performed with 12 E. coli colonies to confirm that all three PCR products were inserted (see FIG. 2B), and one of them cloned (marked with red box) was sequenced. As a result, it was confirmed that the correct nucleotide sequence was cloned (see SEQ ID NO: 1).

Example  3: Arabidopsis transformation of seed-specific promoters

The pBI101 vector used for cloning the At5g54000 promoter (EI promoter) has a GUS (β-glucuronidase) reporter gene between the left and right boards, and is a binary vector that can be transformed into plants. (See FIG. 3).

This vector was transformed into Agrobacterium (GV3101) and transformed into Arabidopsis by the method of flower dipping (Clougher et al., Plant J., 16 (6): 735 743, 1998) Respectively. Transgenic Arabidopsis thaliana was selected on a medium containing the antibiotic kanamycin.

Example  4: Histochemical staining analysis of transformed Arabidopsis thaliana

GUS activity from each tissue of selected transgenic plants was examined by histochemical staining. Each tissue except for the seeds contained 1 mM X-Gluc (5-bromo-4-chloro-3-indoly-β-glucuronidase), 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 at 37 ° C. for 24 hours. Then, the solution was removed, and 70% ethanol was added thereto to remove chlorophyll for 2 hours. This procedure was repeated three times , Followed by storage in 100% ethanol. As a result, GUS staining was not observed in all tissues (roots, stems, leaves, flowers) except seeds as shown in FIG. 4A. This result indicates that the At5g54000 gene promoter (EI promoter) has no promoter activity in tissues such as roots, stems, leaves and flowers.

On the other hand, the seed of the transgenic plant was reacted for 24 hours at 37 ° C. in an aqueous solution containing X-Gluc (5-bromo-4-chloro-3-indoly-β-glucuronidase), a substrate of the GUS gene, and then the solution was After the removal, the solution was first mixed with ethanol and acetic acid at a ratio of 9: 1, and the pigment was first removed at 25 ° C. for 24 hours, and then washed twice with 90% ethanol for 30 minutes. After removing the dye for 24 h at 25 ° C with 1 ml of 30% glycerol and 2.5 g of chloral hydrate, the activity of GUS was observed under an optical microscope (Olympus model BX-51) using a Nomarski lens Respectively. As a result, GUS staining was specifically confirmed in the embryo tissue and the seed endothelium inside the seed (see FIGS. 4A to 4C). In immature seeds, GUS staining was observed only in the endothelium, and it was confirmed that GUS staining was very strong from the time when embryos grew to form cotyledon (see FIGS. 4B and 4C). In addition, cross-sectional GUS staining of mature seeds was confirmed to be strongly stained only in the embryo tissue and the inner integument tissue excluding the outer cortex (see FIG. 4C). These results indicate that the EI promoter exhibits strong promoter activity specifically for embryo tissue and inner integument.

Attach an electronic file to a sequence list

Claims (7)

Embryo tissue and seed endothelial specific promoters of isolated plant seeds having the nucleotide sequence of SEQ ID NO: 1. The promoter according to claim 1, wherein the plant is a dicotyledonous plant. An expression vector for transformation comprising the promoter of claim 1. An Escherichia coli comprising the expression vector for transformation according to claim 3. A plant transformed with the expression vector of claim 3. 1) transforming a plant with a recombinant expression vector comprising the promoter of claim 1 and a gene encoding a target protein; And
2) A method for producing a target protein comprising the step of expressing a gene encoding the target protein in the embryo tissue and seed endothelial of the transformed plant seed to produce the target protein. A primer for PCR as set forth in SEQ ID NO: 4 and SEQ ID NO: 5 for amplifying the promoter of claim 1.

Images