KR20100092295A - Flower and fruit specific expression promoter from solanum lycopersicum hr7 gene and uses thereof - Google Patents

Abstract

PURPOSE: An expression promoter which is specific to plant and fruits derived from Solanum lycopersicum HR7 gene is provided to develop transgenic plant. CONSTITUTION: An expression promoter which is specific to plant flower and fruit contains 1-890 base sequence of sequence number 1. 5'-UTR(untranslated region) contains 891-966 base sequence of sequence number 1. A plant expression vector contains the expression promoter, 5'-UTR, or mixture thereof. The plant expression vector is pCAM-1391HR or pCAM-2300HR. A method for expressing a foreign gene in the transgenic plant by flower and fruit-specificity comprises: a step of recombining the foreign gene to a plant expression vector; and a step of transforming the recombinant plant expression vector to the plant.

Description

Flower and fruit specific expression promoter from Solanum lycopersicum HR7 gene and uses according to tomato HR7 gene

The present invention relates to a tomato HR7 gene-derived flower and fruit specific expression promoter and its use, and more particularly to a tomato HR7 gene-derived plant flower and fruit specific expression promoter and a 5 'untranslated region (hereinafter referred to as 5'-). Abbreviated as UTR), a flower and fruit specific expression vector comprising the same, a plant transformed with the expression vector, a method for expressing a flower and fruit specificly with a foreign gene using the expression vector, and a foreign by the method. The present invention relates to a transgenic plant in which genes are specifically expressed in flower and fruit and seeds thereof.

The HR7 gene, like the metallocarboxypeptidase inhibitor (MCPI), is a new protein containing the DUF1532 superfamily domain, which consists of about 80 amino acids. The gene was first cloned from the root hairs through the root hair cDNA library of Hyoscyamus niger and specifically expressed in the basal part of the root hair roots and primitive cells. Metallocarboxypeptidase inhibitors (MCPIs) that bind to metallocarboxypeptidase have been found in eggplant plants such as tomatoes and potatoes (Liu et al. (2000). Mol. Biol. Rep., 27: 241-246), and Hirudo medicinalis , It has also been found in pig carcasses ( Ascaris suum ) , Rhipicephalus bursa, rats and human tissues. The MCPI of a plant is a small peptide of about 10 KDa consisting of 90-110 amino acid residues. These MCPIs are exceptionally competitively potent in inhibiting a wide range of animal and microbial carboxypeptidases except for yeast and plant serine carboxypeptidase (Hass GM, Hermodson MA. (1981) Biochemistry, 20: 2256-2260). MCPI was found to accumulate in tuber and leaf tissues during potato development, and MCPIs from other families responded to wounds and were more highly expressed in fruit than in leaves (Ryan CA. (1990)). Annu. Rev. Phytopathol, 28: 425-449). In addition, HR7 gene of Capsicum annuum cv. Bukang is mainly expressed in the EST library made from seeds, so it is presumed that it is specifically expressed in seeds or is involved in seed formation during the development of pepper (http: // sol. pdrc.re.kr/), its function or role has not been revealed to date.

Methods of analyzing gene function at developmental stages or at specific organs can be reduced to three levels: The first is to overexpress the gene to make a mutant that can be identified at each stage of development or by observation of specific tissues. Or to find a phenotype that disappears by suppressing the gene. The second is to identify the level of transcription of the gene. Currently, the method focuses on the actual expression level of mRNA by screening cDNA libraries using real-time PCR or microarray data. The third is promoter trapping. This method finds promoters of specific genes and links them with marker genes to identify their expression. In this method, genes expressed only in specific situations or in specific tissues can be more easily identified through promoters. Theoretically, any developmental stage can be applied and has the advantage of not being studied depending on mutations or natural phenotypes (Belinda et al. (1991) Molecular and General Genetics MGG, 228: 281-286).

In plants, the promoter region is located from 1 to 2 kb, up to 3 kb, towards the 5'-upsteam of the gene, and the promoter regulates expression by initiating transcription of the gene. In addition, unlike in prokaryotic cells, in eukaryotic cells, one promoter regulates a single gene, and thus, genes that are expressed according to a specific physical and chemical situation, or which are developed or tissue-specifically expressed are searched for. It is easy to develop and can use it. However, as of today, since the function is not known or a few genes are isolated, the mechanism of the promoter with a specific expression pattern in the development of the plant is difficult to understand genetically.

Methods for studying such promoter activity include a system for making a transgenic plant through experiments with Agrobacterium-mediated transformation method that induces stable gene expression, and a method for quick and easy analysis through transient expression analysis using Agroinfiltration. Transgenic plants produced through the transformation method using Agrobacterium may be a waste of time and labor as well as cost and labor due to different culture conditions for each plant species. However, the transient expression system using the agroinfiltration method saves more labor, and thus saves time in analyzing as well as cost. In addition, it can be used for various plant species such as tobacco and Arabidopsis as well as tomato, and agroinfiltration method can be used to economically perform experiments on not only expression of specific genes but also promoter activity (Yang et al. (2000) The Plant J. 22 (6): 543-551).

Korean Patent Registration No. 0784165 discloses a promoter for controlling UDP-glucosyltransferase derived from pepper plants involved in the pathogenic infection of pepper plants and the ripening process of pepper berries, and in Korean Patent Registration No. 0574563 Arabidopsis-derived plant high efficiency expression promoter and a plant high efficiency expression vector containing the same are disclosed, but different from the promoter of the present invention.

The present invention has been made in accordance with the requirements as described above, after cloning the promoter and 5'-UTR of tomato HR7 gene, inserting the promoter and 5'-UTR in a binary vector and introduced into the Arabidopsis and tomato, The present invention has been completed by revealing that foreign genes are specifically expressed in flower and fruit tissues of transformed plants.

In order to solve the above problems, the present invention provides a flower and fruit specific expression promoter or 5'-UTR derived from HR7 gene of tomato.

The present invention also provides a flower and fruit specific expression vector and a plant transformed with the expression vector comprising the flower and fruit specific expression promoter and / or 5'-UTR.

In addition, the present invention provides a method for expressing a foreign gene in plant flowers and fruits using the flower and fruit specific expression promoter or 5'-UTR, if the mass production of useful substances in plant flowers and fruits.

The present invention also provides a transgenic plant and seeds thereof, wherein the foreign genes produced by the method are specifically expressed in flower and fruit.

According to the present invention, as a result of expressing a foreign gene in a plant using a flower and fruit specific expression promoter derived from HR7 gene of tomato and 5'-UTR, the gene can be specifically expressed in plant flower and fruit. It was confirmed that it is a new promoter.

In order to achieve the object of the present invention, the present invention provides a plant flower and fruit specific expression comprising the nucleotide sequence of 1 to 890 (-890 to -1 site from the transcription initiation site) of the sequence (SEQ ID NO: 1) of FIG. Provide a promoter.

While the CaMV35S promoter derived from Cauliflower mosaic virus, which is widely used, expresses a gene introduced from all tissues, the flower and fruit specific expression promoter of the present invention is a flower and fruit of a gene introduced from a transgenic plant. Can be expressed specifically.

In order to achieve the object of the present invention, the present invention provides a 5'-UTR comprising a nucleotide sequence of 891 to 966 (+1 to +76 sites from the transcription initiation site) of the sequence of Figure 3 (SEQ ID NO: 1). .

In addition, variants of such promoter sequences or 5′-UTR sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in base sequence but has similar functional properties to that of SEQ ID NO: 1. Specifically, the promoter sequence and the 5'-UTR sequence are at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 1 It may include base sequences having homology.

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).

In order to achieve another object of the present invention, the present invention provides a flower and fruit specific expression vector comprising a flower and fruit specific expression promoter and / or 5'-UTR of the plant.

The flower and fruit specific expression vector of the present invention may contain only the promoter of the present invention or may be used in combination with a general plant expression promoter such as a CaMV 35S promoter in the 5'-UTR of the present invention, but preferably the present invention. It is good to include both the promoter and 5'-UTR of the gene for the expression of flowers and fruits specifically in the plant.

The flower and fruit specific expression vector of the present invention can be used as a transient expression vector capable of temporarily expressing in a plant into which a foreign gene has been introduced and a plant expression vector capable of permanently expressing a foreign gene in a introduced plant. Can be.

Binary vectors that can be used in the present invention may be any binary vector containing the right border (RB) and left border (LB) of T-DNA capable of transforming plants in the presence of Ti plasmid of A. tumefaciens . Preferably, pBI101 (Cat #: 6018-1, Clontech, USA), pBIN19 (Genbank Accession No. U09365), pBI121, pCAMBIA vectors, and the like, which are frequently used in the art, may be used.

Flower and fruit specific expression vector according to an embodiment of the present invention may be pCAM-1391HR or pCAM-2300HR shown in Figure 1, but is not limited thereto. The promoter of the present invention was inserted into a binary vector for promoter analysis (pCAMBIA 1391Z) having a reporter gene to prepare pCAM-1391HR or pCAM-2300HR (FIG. 1), and was used for plant transformation using Agrobacterium ( Agrobacterium ). It will be apparent to those skilled in the art that the reporter gene may be substituted with another desired foreign gene.

The term “vector” is used when referring to DNA fragment (s), nucleic acid molecules that are delivered into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. 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 a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the genes 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 preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by a chemical method, which corresponds to all genes capable of distinguishing transformed cells from non-transformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol It doesn't happen.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, such as nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminator of the octopine gene of Bacterium tumerfaciens ( Agrobacterium tumefaciens ), but is not limited thereto.

In order to achieve the further object of the present invention, the present invention provides the flower and fruit of the plant-specific E. coli transformed with the expression vector of the invention (E. coli) or Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens).

In order to achieve another object of the present invention, the present invention provides plants and seeds thereof transformed with the flower and fruit specific plant expression vector of the present invention.

The flower and fruit specific plant expression vector of the present invention can transform any plant irrespective of dicotyledonous or monocotyledonous plants, but in the present invention, transformation was performed in Arabidopsis and tomato. The plant according to an embodiment of the present invention is tomato, Arabidopsis, potato, eggplant, tobacco, pepper, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, It may be a dicotyledonous plant, such as gat, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous 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 tumerfassi by plant infiltration or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises 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.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

"Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, fruits, 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.

In order to achieve another object of the present invention, the present invention comprises the steps of recombining a foreign gene to the flower and fruit specific expression vector of the present invention; And

Provided is a method of expressing a flower and fruit specificly in a transgenic plant, wherein the foreign gene comprising the step of transforming the recombinant flower and fruit specific expression vector into a plant.

The foreign gene may be any gene desired to be expressed in plant flowers and fruits, and may be located after the promoter in the flower and fruit specific expression vector of the present invention and may be expressed by fusion with a reporter gene as necessary. The recombinant flower and fruit specific expression vector may be transformed into a plant as described above. In the method according to an embodiment of the present invention, the plant is tomato, baby pole, potato, eggplant, tobacco, pepper, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage And dicotyledonous plants, such as cabbage, mustard, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, or pea.

In order to achieve another object of the present invention, the present invention provides a transgenic plant and seeds thereof, wherein the foreign gene produced by the method is specifically expressed in flower and fruit. The transgenic plant can express flower and fruit specific foreign genes by flower and fruit specific expression promoter and / or 5'-UTR.

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

Materials and methods

1. Plant materials and culture conditions

Tomato plant ( Solanum lycopersicum cv.Micro-Tom) was maintained at a temperature of 24 ℃ in the culture chamber, Arabidopsis thaliana ecotype columbia-0 The plant was kept constant at a temperature of 20 ° C. The daylight cycle gave 16 hours of light and 8 hours of dark.

2. Separation of tomato genomic DNA and genomic walking PCR to identify 5'-upstream regions

To extract tomato genomic DNA, the tomato leaf tissue was finely ground using liquid nitrogen, and then extracted according to the protocol of DNeasy plant mini kit (Qiagen, Germany). Thereafter, 2.5 ug of genomic DNA was treated with 80 units of Dra I , EcoR V , Pvu II , Stu I for 16 hours at 37 ° C. to form blunt ends, and purified with phenol: chloroform (1: 1). After recovering with 100% ethanol was dissolved in 20ul of sterile distilled water. Of these, 4ul was ligated with Genome walker Adaptor fragment (clontech, USA), and primary PCR was performed with Adaptor Primer 1 (AP1) and HR7 Gene Specific Primer 1 (GSP1), and the reaction composition was as follows: DNA Polymerase buffer, 1.5 mM Mg (OAc) ₂ , 2.5 mM dNTPs (dATP, dTTP, dCTP, dGTP), respectively, 10 pM primers (AP1 and HR7 GSP1); 1ul ligated DNA, 0.1U DNA polymerase. In addition, the first PCR reaction conditions are 25 seconds at 94 ℃; After 7 cycles at 72 ° C. for 3 minutes, 94 ° C., 25 seconds; 32 cycles were performed at 67 ° C. for 3 minutes. Further, 67 ° C. was performed once for 7 minutes, and the reaction was terminated by lowering to 4 ° C. The primary PCR product was diluted 1/50 to perform nested secondary PCR with AP2 and GSP2. The reaction composition was performed as the primary PCR reaction composition, and the reaction of nested secondary PCR was 94 ° C. for 25 seconds; After 5 cycles at 72 ° C. for 3 minutes, 94 ° C., 25 seconds; 20 cycles were performed at 67 ° C. for 3 minutes, and 67 ° C. was performed once for 7 minutes to lower the reaction to 4 ° C. to terminate the reaction. Primer sequences used in the above PCR are shown in Table 1. Electrophoresis was performed on 1% agarose gel to confirm the PCR result.

Table 1. Primer designs for cloning the SlHR7 promoter region. HR7 GSP1, nested HR7 GSP2, AP1, nested AP2 primers were used to isolate the 5'- flanking region of the SlHR7 gene by genomic walking PCR, and the HR7 F, HR7 R, H700F, H350F primers were used for transgenic Arabidopsis and It was used to prepare constructs for transient expression analysis in wild type tomatoes (GSP, gene specific primers; AP, adapter primers).

* Underlined parts show restriction enzyme sites.

3. DH5α / E.coli  Preparation and transformation of competent cell (CP cell)

E. coli strain DH5α was inoculated in 3 ml of LB medium (peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH7.2) and shake-cultured at 37 ° C. for 18 hours or more, and then passaged in 100 ml LB liquid medium. The culture was incubated with an OD value of 0.4 to 0.45 at 600 nm. The culture was left on ice for 15 minutes, and then recovered by centrifugation at 4000 rpm for 20 minutes at 4 ° C. The recovered E. coli was treated with ice-cold 80 mM MgCl ₂ -20 mM CaCl ₂ solution. This was placed on ice for 15 minutes, centrifuged at 4000 rpm / 4 ° C. for 20 minutes, treated with ₂ ml of ice-cold 0.1M CaCl ₂ solution, and used for transformation. In case of immediate use, it was used after being put on ice for 30 minutes. Otherwise, 15-20% glycerol was added and stored at -70 ° C.

After mixing the DNA with the qualified cells, and placed on ice for 30 minutes or more, and then subjected to heat shock by leaving for 90 seconds at 42 ℃, 1 ml of LB liquid medium containing no antibiotics was added and incubated for 1 hour at 37 ℃. . Subsequently, transformed colonies were selected by plating on LB medium containing antibiotics. Selected colonies were cultured to accup prep. The plasmid was isolated using a mini kit (Bioneer, Korea) and confirmed using restriction enzymes.

4. Agrobacterium Agrobacterium Eligible Cell Preparation and Transformation

Agrobacterium was inoculated in 5 ml of YEP (10 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl, pH7.2) liquid medium and shake-cultured at 28 ° C., and the cultured 1 ml was transferred to 50 ml YEP liquid medium. Incubated at 600 nm until the OD value is 0.6 ~ 1.0. This was placed on ice for 30 minutes, centrifuged at 4000 rpm / 4 ° C. for 20 minutes, resuspended in ice-cold 0.15M NaCl, and then placed on ice for 10 minutes. Thereafter, the pellets recovered by centrifugation at 4000 rpm / 4 ° C. for 20 minutes were resuspended in 1 ml of 20 mM CaCl ₂ and dispensed in 50ul portions. It was quenched in liquid nitrogen and stored at -70 ° C.

1ug of DNA was stored in the stored qualified cells, and then frozen in liquid nitrogen for 2 minutes, heat shock was applied at 37 ° C. for 5 minutes, lightly mixed, and the above procedure was repeated once. After 30 minutes on ice, 1 ml of YEP liquid medium was added, incubated at 28 ° C. for 2 hours, and then plated on YEP solid medium containing Rif 50 mg / L and Km 50 mg / L to transform the colonies. Screened.

5. SlHR7 Recombinant vector at the promoter site

The SlHR7 promoter site identified 1 kb region by genomic walking PCR, which was then PCR with HR7 F primer and HR7 R primer, cut with HindIII and BamHI enzymes for recombination into pCAMBIA1391Z, followed by T4 ligase 3 at 24 ° C. Time ligated, DH5α / E. coli eligible cells were transformed, selected from LB + Km 50 mg / L agar plate and confirmed by cutting with Hind III and BamH I enzyme, plasmid was extracted. The extracted plasmids were transformed into Agrobacterium C58C1 strain qualified cells and selected from YEP + Rif 50mg / L + Km 50mg / L agar plates, which were used for plant transformation using Arabidopsis.

In order to perform tomato transient expression assay, the SlHR7 promoter region was recombined to the vector fused to pCAMBIA2300 with GFP and transformed into Agrobacterium strain LBA4404 qualified cells. In addition, to confirm the role and expression level of the cis-acting element, to perform the deletion test, the SlHR7 promoter region was recombined into -700 / -1 region and -350 / -1 region, respectively. First, each site was PCR-recombined with H7 F primer and H700 F primer and H350 F primer at full length, cut with Hind III and BamH I enzymes, and recombined into pCAMBIA2300-GFP vector, which was then subjected to Agrobacterium strain LBA4404 qualified cells. Was transformed. Primer sequences used for cloning are shown in Table 1.

6. Arabidopsis transformation by vacuum-infiltration method experiment

Arabidopsis thaliana ecotype columbia-0 was used in Arabidopsis thaliana ecotype columbia-0 and was grown for about 4-6 weeks in soils with vermiculite: pearlite: pitmos (2: 2: 1).

The Agrobacterium C58C1 strain transformed with the recombinant vector pCAM-1391HR was preincubated at 28 ° C., incubated for 24 hours in 120 ml of YEP liquid medium containing antibiotics, and centrifuged at 4500 rpm / 4 ° C. for 20 minutes to recover the cells. VIF [vacuum infiltraion medium, MS salts with B5 vitamin 2.2 g / L (Duchefa biochemie, USA), 5% sucrose, MES 0.5 g / L, pH5.7 with KOH, BAP 0.044 uM, silwet L-77 200 ul / L ), And the cells were adjusted to OD> 2.0. Agrobacterium Floating Prepared Arabidopsis Plants Turn the flower organ down on the beaker containing the cell solution and turn it upside down so that all can be locked, and maintain the vacuum of 4-10pa for 15-20 minutes. Thereafter, the vacuum was quickly released, sealed in a bag to allow one day of purification, and water was supplied. The transformed Arabidopsis seeds were disinfected with 70% ethanol and 10% sodium hypochlorite solution and washed with sterile distilled water to remove 1/2 MS agar containing 200 mg / L cytocecsim and 35 mg / L hygromycin. Selected on plates (2.2 g / L MS, 2% sucrose, pH5.8, 0.8% agar powder) (Bechtold, Ellis and Pelletier (1993) CR Acad. Sci. Paris, Life Sciences 316: 1194-1199).

7. Histochemical GUS Assay and GUS Activity Measurement

Histochemical GUS assay was performed to confirm the expression pattern in the transformed Arabidopsis. The leaves, flowers, stems, roots and seed tissues of the transformed Arabidopsis plants were GUS stained [100 mM sodium phosphate buffer (pH 7.2) 10 mM EDTA (pH 8.0), 0.5 mM K ₂ Fe (CN) ₆ , 3 % x-Glc A (Duchefa biochemie, USA)], and placed at 37 ℃ for about 24 hours, and fixed with 100% ethanol.

GUS can hydrolyze a fluorescent substrate called 4-MUG (4-methylumbeliferryl-β-D-glucuronide) with 4-MU (Methylumbeliferone). GUS activity was measured based on 4-MU hydrolyzed during the reaction. . GUS To measure activity, each plant tissue was first frozen in liquid nitrogen and ground finely with a pestle, followed by GUS extraction buffer [50 mM KPO (pH 7.0), 0.1 M EDTA (pH8.0), 1% Triton X-100, β-mercaptoethanol] and then 10ul of the sample was pre-warming 190ul GUS assay buffer [100 mM KPO ₄ (pH 7.2), 0.1M EDTA (pH8.0), 1% Triton X-100, β-mercaptoethanol , MUG 22mg / 25ml)], and after exactly 30 minutes at 37 ℃, the reaction was terminated by the addition of 800ul stop buffer (0.2M NaCl). This was measured for fluorescence of 360 / 460nm. The measured value was quantified by BSA (albumin serum bovine, sigma, USA) by diluting 5ｘ Bradford reagent (50 ml of 95% ethanol, 100 ml of 85% H ₃ PO ₄ , 0.1 g Briliant blue, 50 ml of water) to 1/5. Nomalization with soluble protein. GUS activity can be expressed in nmol4-MU · min ⁻¹ ug ⁻¹ protein units.

8. Transient Expression Analysis

In order to perform transient expression analysis, Agrobacterium LBA4404 strain was pre-cultured, passaged in 20 ml YEP liquid medium containing 200 uM acetosyringone, grown to OD = ~ 0.7, and centrifuged at 4500 rpm / 4 ° C for 20 minutes to recover the cells. Resuspend in 20 ml of 1 / 2MS medium (MS 2.2g, 2% sucrose, pH5.7) with 200uM acetosyringone and infect it with Agrobacterium in a tomato seed with a syringe and infiltrate leaf and fruit tissues (Agroinfiltration ). Samples were taken 2.5 days later, and then, with the protein extraction buffer (PEB), and prepared by centrifugation at 15000 rpm / 4 ℃ for 30 minutes, the expression level was confirmed by ELISA method using His tag monoclonal antibody, Agrobacterium Normalization was based on the amount of GUS expression expressed in the bacteria.

9.Enzyme-linked immunosorbent assay (ELISA)

First, the sample was ground with protein coating buffer (PCB, 3.3 g / L Na ₂ CO ₃ , 6.0 g / L NaHCO ₃ , pH9.6), and then centrifuged at 15000 rpm at 4 ° C. to supernatant the 96-well microplate. 100ul was dispensed into and placed at room temperature for 2 hours. Then, washed with protein extraction buffer (PEB, 1.16g / L Na ₂ HPO ₄ , 0.1g / L KCl, 0.1g / LK ₃ PO ₄ , 4.0g / L NaCl, pH7.4), blocking buffer (5 % skim milk) 200ul was dispensed and placed at room temperature for 2 hours. Then, after washing with PEB twice, 50ul of primary antibody (novagen, Germeny) diluted to 10 ^-7 was dispensed at room temperature for 2 hours to allow interaction between the desired proteins. . Thereafter, after washing four times or more with PEB, the secondary His tag monoclonal antibody (sigma, USA) was diluted with 1: 2000 PEB and 50ul was dispensed. The reaction was allowed to stand at room temperature for 1 hour, washed four times with PEB, and then 50 μl of substrate solution (BD bioscience, USA) was dispensed and left at room temperature for 30 minutes, followed by 50 μl of stop solution (2.5MH ₂ SO ₄ ). Dispense to terminate the reaction. This value was measured by absorbance at 450 nm (Engvall E, Perlman P (1971) Immunochemistry 8 (9): 871-4).

Example 1. SlHR7  Find the 5'-upstream region of the gene

After extracting the tomato genomic DNA to find the promoter of the tomato HR7 gene ( SlHR7 ), it is cut with each of the EcoR V, Dra I, Pvu II and Stu I restriction enzymes to produce blunt ends. Was prepared to prepare tomato genomic DNA libraries. Genome walking PCR was performed using HR7 GSP1 and GSP2 primers located in the exon portions of AP1 and SlHR7 , and a 1.2 kb DNA band was identified in tomato genomic DNA libraries made from EcoR V restriction enzymes (FIG. 2).

Cloning this into the pGEM-T easy vector (Promega, USA) and confirming the nucleotide sequence, the N-terminal region of the SlHR7 gene, 76 bp 5'-untranslated region (UTR), about 1 kb promoter region was confirmed. The nucleotide sequence of the SlHR7 promoter region was analyzed by the PLANTCARE (www.plantcare.com) system. As a result, the nontranslated region (UTR) was present 76nt before the translational start site ATG as the 5'-upstream of the SlHR7 gene. The starting cytidine (C) base was marked with +1 nt and various cis-acting regulatory elements were found (FIG. 3).

The minimal promoter site is estimated to be the CAAT box and TATA box at -112nt and -54nt, respectively.In addition to the cis-acting elements on the base sequence of the SlHR7 promoter, a GA motif was found at -167nt and an AAGAA motif at -178nt. It is confirmed to exist. It was also confirmed that AT rich elements were found in -335nt and -590nt, ARE was found in -474nt, and G-box was found in -513nt. TATCCAT / C motif was identified in -794nt of the SlHR7 promoter region, and ACE was found in -800nt (Fig. 3).

Example 2 . Expression and Activity Analysis of SlHR7 Promoter in Transgenic Arabidopsis Plants

The 5 'flanking region of the SlHR7 gene determined by genome working PCR was PCR amplified with HR7 F / R primer set to identify the nucleotide sequence, using only the SlHR7 promoter region including the 5' UTR region, and then the BamH I and PCMA-1391HR was prepared by recombination with the plant promoter analysis binary vector pCAMBIA1391Z (http://www.cambia.org) cut into Hind III (FIG. 1).

Recombinant pCAM-1391HR was transformed into Agrobacterium tumefacience C58C1, and then transformed into Arabidopsis thaliana ecotype columbia-0 using a vacuum-infiltration test method to induce stable gene expression, and recovered T _1. Arabidopsis seeds of the generation were screened with 35 mg / L of hygromycin and subjected to histochemical GUS analysis to confirm expression of the SlHR7 promoter.

Histochemical GUS analysis revealed insoluble blue by reacting with β-glucuronidase (GUS) -expressing tissue, X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide). The expression of the GUS gene can easily identify the expression of the promoter. First, after collecting each tissue, the tissue was immersed in the GUS staining solution, overnight at 37 ℃, and fixed with 100% ethanol.

As a result, GUS gene was expressed nonspecifically in leaves, roots, stems, and flower organs, and similar expression was observed in stem tissue, leaf tissues, and root tissues compared to positive control 35S promoter, but only in the surgical part in flower organs. Unlike the 35S promoter, the SlHR7 promoter was confirmed to be expressed higher in the pistil and calyx parts as well as the surgery (FIG. 4).

In addition, to confirm the actual expression level in each tissue, GUS The activity was confirmed. As a result, the expression of leaves, seeds, and stem tissues was similar to that of the 35S promoter, but in flower tissues, the expression was more than 2.5 times higher than that of 35S, especially in flowers compared to other tissues (Fig. 5).

Example 3 . Activity Analysis of SlHR7 Promoter in Tomato

To confirm the expression of SlHR7 promoter in tomato, Agrobacterium -Infiltration method (Agroinfiltration) was used. First, in order to use the marker GFP by PCR for GFP gene site of pCAMBIA1302 vector cut with BamH Ⅰ and EcoR Ⅰ, by recombination in pCAMBIA2300 vector pCAMBIA-GFP we created a vector, by PCR the SlHR7 promoter region Hind Ⅲ and BamH Ⅰ After cleavage, the vector was recombined into pCAMBIA2300-GFP vector to generate a vector pCMA-2300HR for transient expression analysis (FIG. 1). This was transformed into Agrobacterium tumefecience LBA4404 and used for tomato transient analysis. Agro-infiltration tissues were collected after 2.5 days, and their expression was confirmed by ELISA using his tag monoclonal antibody. In addition, since the GUS gene is expressed in Agrobacterium bacteria itself, it was used to quantify it.

As a result of transient expression analysis, tomato seed and leaf tissues showed lower expression levels than the 35S promoter, but tomato tea tree fruit showed about 2 times higher expression levels than the 35S promoter, which was about 1.3 times higher than the leaf tissue. Indicated. Therefore, it was confirmed that the fruit specific expression of the SlHR7 promoter (Fig. 6).

Example 4. Investigation of promoter active sites using deletion assay

In order to identify the active site on the promoter sequence and search for the cis-acting element necessary for expression, a 5'-deletion analysis test of the SlHR7 promoter was performed. First SlHR7 promoter approximately 1kb in size of full-length promoter region (-890 / + 76, FL) were amplified in a HR7 F / R primer, the whole of the promoter 5'upstream region of about 270bp is removed Hd1 construct (-619 / + 76 ) Was recombined into the pCAMBIA2300- GFP vector by PCR with Hd1 F / HR7 R primer, construct Hd2 (-262 / + 76) from which approximately 630 bp of the 5'upstream site was removed with Hd2 primer. This was transformed into Agrobacterium strain LBA4404, respectively, and then transient expression analysis was performed on tomato leaf tissues.

As a result, promoter activity was the highest in construct FL (-890 / + 76), and the expression levels of construct Hd1 and construct Hd2 gradually decreased, whereas construct FL and Hd1 showed a 61% decrease, and construct Hd2 And showed a 73% difference in expression rate. Construct FL (-890 / + 76) of the SlHR7 promoter showed higher promoter activity (FIG. 7), indicating that the cis-acting element affecting promoter activity was included in the -890 ~ 619 region of the SlHR7 promoter. do.

1 is a schematic diagram of promoter analysis vectors for Arabidopsis transformation and tomato transient analysis. pCAM-1391HR is a Arabidopsis transformation vector and pCAM-2300HR is a transient expression vector in tomato. HR7, tomato HR7 promoter; HYG (R), hygromycin resistance gene; GUSA, β-glucuronidase A coding gene; tNOS, NOS terminator; NPTII, kanamycin resistance gene; CaMV 35S, cauliflower mosaic virus 35s promoter; GFP gene, green fluorescent protein coding gene; His tag, hexa histidine tail; LB, left border; RB, right border.

2 shows genomic walking PCR to isolate the SlHR7 promoter region. Tomato genome libraries were digested with EcoR V, Dra I, Pvu II, and Stu I, respectively , and then ligated with adapter fragments to separate the 5'- flanking regions of the SlHR7 gene by genome working PCR (M, size marker: 1, tomato genomic DNA library by EcoR V: 2, Dra I: 3, Pvu II; 4, Stu I).

Figure 3 shows the SlHR7 promoter sequence of the 5 'flanking region of the upstream of ATG start codon. nt's were numbered starting from C (+1) of 5′-UTR. Virtual CAAT box at -112 position, TATA box at -54 position, and transcription start site at +1 position. There are cis-acting elements: GA motif (-167nt), AAGAA motif (-264nt), AT rich elements (-335nt and -590nt), ARE (-474nt), G-box (-513nt), TATCCAT / C motif (-794nt), ACE (-800nt).

4 is Histochemical staining results for tissues of Arabidopsis plants transformed with pCAM-1391HR and pCAMBIA1301 (WT, wild type; 35S, CaMV 35S promoter; SlHR7 , HR7 promoter).

Figure 5 GUS expression levels for tissues of Arabidopsis plants transformed with pCAM-1391HR ( SlHR7 promoter) and pCAMBIA1301 (35S promoter). It was measured in each tissue of the transgenic Arabidopsis. GUS activity on the reporter gene of the target promoter was normalized to total protein by Bradford reagent (35S as a positive control, CaMV 35S promoter / pCAMBIA1301; H, SlHR7 promoter / pCAMBIA1391Z).

6 shows a transient analysis of wild type tomato seeds, leaves and berries. The transient assay was performed by agroinfiltration and the expression level of GFP was measured by ELISA using his-tag monoclonal antibody with 450 nm absorbance. Values were normalized by GUS activity (GFP, Green Fluorescent Protein; TSP, Total Soluble Protein; mg, tomato green stage).

7 shows transient GFP expression for full length and deletion of the SlHR7 promoter in tomato. Deletion analysis was performed by agro-infiltration and the expression level of GFP was measured by ELISA using his-tag monoclonal antibody with 450 nm absorbance. Values were normalized by GUS activity (GFP, Green Fluorescent Protein; TSP, Total Soluble Protein; mg, tomato green stage).

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> Flower and fruit specific expression promoter from Solanum          lycopersicum HR7 gene and uses young <130> PN09002 <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 966 <212> DNA <213> Solanum lycopersicum <400> 1 tgtgaatact atccaggatt tactatcata tccgacccct ctaacttatc ccggtccgac 60 ctatgaccgt ttgtaaatga actacgatgg tatccatttt tcctgttctt ttcacaagtg 120 attattttta ctttcctcac ttattcttgg ctatagcact tacgtatatg ttacaaatct 180 taacattgat ttttttctga ggatcttcaa tcaattgaaa cggttttggc ttttgcacac 240 aataatgttt tttcaatatt gtctttttaa tgatgaatta agaatggtca taaaaatcaa 300 ttgtggatac catgtaggct tgattatata tttaatgcat cagtatttta ctaatctatt 360 taattagcac acatggatta gttgtttttg ggaaacttag gatcgttagt ttggtttctc 420 tttcttttgg ccttattttg attgcaatct tagcctattt ctttggaaat tatccaccat 480 tgattattgt tatagccctc aactaattga ctattttacc actctacata aattattttt 540 atagccttca attaattaaa tctaagttat tgatacaata ttagacatat gaaaattgac 600 attcattcga gaatatgttg aaaaataaga atggtaggta aatccttttt ttacaaaaat 660 atatttctct ttacactata acaattttcc cctaaataat aaggtaaaaa aaatgtaaat 720 atagataaaa taaaaataat agaactcaca caacatattg tacaattaag tactacaatg 780 ttagtccttt ttgttttagg cttcacaaat aatgcttcat aaaatatctt tattttaaaa 840 aaccaaaaaa aatatgaacc aagtgtagta atatgatcct ataaatacaa cattggttga 900 gaaggaaatt cacccttcac atattcttct tttttccatt ttccaatcaa agtagcatca 960 cataat 966 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> HR223 GSP 1 primer <400> 2 acccattgct tgcatctttg gtgaacg 27 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Nested HR7 GSP 2 primer <400> 3 tgcatctttg gtgaacggga catggta 27 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AP1 primer <400> 4 gtaatacgac tcactatagg gc 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nested AP2 primer <400> 5 actatagggc acgcgtggt 19 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> HR223 F primer <400> 6 ctaagctttg tgaatactat ccaggatt 28 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> HR223 R primer <400> 7 gtggatccat tatgtgatgc tactttgatt 30 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> H223 F primer <400> 8 tcaagcttga tgaattaaga atggtca 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> H223 F primer <400> 9 tcaagcttga atggtaggta aatcctt 27  

Claims (10)

Plant flower and fruit specific expression promoter comprising the nucleotide sequence of 1 to 890 (-890 to -1 site) of SEQ ID NO: 1. 5′-UTR comprising nucleotide sequences of 891 to 966 (+1 to +76 site) of SEQ ID NO: 1. A plant expression vector comprising the promoter of claim 1, the 5′-UTR of claim 2, or the promoter of claim 1, and the 5′-UTR of claim 2. The plant expression vector according to claim 3, which is pCAM-1391HR or pCAM-2300HR shown in FIG. The third or claim 4 of the plant expression vector transformed with Agrobacterium tyumeo Pacific Enschede (Agrobacterium tumefaciens). A plant transformed with the plant expression vector of claim 3. 7. The transformed plant according to claim 6, wherein the plant is a dicotyledonous plant. Recombining the foreign gene into the plant expression vector of claim 3; And A method of expressing flowers and fruits in a transgenic plant, wherein the foreign gene comprising the step of transforming the recombinant plant expression vector to a plant. The method of claim 8, wherein the plant is a dicotyledonous plant. A transgenic plant in which a foreign gene produced by the method of claim 8 is specifically expressed in flower and fruit.

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