KR100859988B1 - Above-Ground Specific Promoter and Above-Ground Specific Expression Method of Target Protein Using the Same - Google Patents

Abstract

The present invention relates to a terrestrial specific promoter and a method of terrestrial specific expression of a target protein using the same. More specifically, the present invention relates to a promoter derived from Arabidopsis s eryngii which can induce the above-ground specific expression of a plant, and a method of expressing the target protein specifically above the above-mentioned plant using the same. The above-ground specific promoter according to the present invention has excellent tissue specificity that can induce the expression of a gene of interest only in the above-ground part of the plant, and thus is useful for the development of a transgenic plant capable of producing a large amount of high value-added useful components. Can be.

Above ground, promoter, transformant, expression vector

Description

Above-Ground Specific Promoter and Above-Ground Specific Expression Method of Target Protein Using the Same}

Figure 1 shows a map of the recombinant vector pBGWFS7-PAtAD5 prepared in one embodiment of the present invention.

<Description of the code>

LB-Left Side

Tnos-nos Terminator

BAR-herbicide resistance gene

Pnos-nos promoter

attB1-Adapter used for gateway cloning

PAtAD5-Terrestrial Specific Promoter of the Invention

attB2-Adapter used for gateway cloning

P35S-Cauliflower Virus 35S Promoter

EGfp-green fluorescent protein gene

GUS-β-glucuronidase Gene

T35S-Cauliflower Virus 35S Terminator

RB-right side

SpR-Spectinomycin Resistance Gene

Figure 2 is a diagram observing the expression of the GUS gene in Arabidopsis transformed with pBGWFS7-PAtAD5 vector of the present invention.

A: Expression of the GUS gene in the cotyledon of seedlings 7 days old germinated in water from Arabidopsis and non-transgenic Arabidopsis transformed with pBGWFS7-PAtAD5 vector of the present invention, respectively.

B: Expression of the GUS gene expression in the ground part of the naïve 4 weeks after germination of the second generation of Arabidopsis larvae transformed with the pBGWFS7-PAtAD5 vector of the present invention,

C: Expression of the GUS gene in the leaves, flowers, stems, pods and roots of the plant 5 weeks after the seedling B was observed. In addition, the numbers 1 to 5 represent each individual of the second generation of transformed Arabidopsis,

D: As a control of C, seeds of the non-transformed Arabidopsis germination were germinated in the soil, and after 5 weeks after the seedlings were observed, the expression of the GUS gene was expressed in the plant body.

Figure 3 after extracting the entire RNA from the leaves, roots, stems and flowers of Arabidopsis transformed with a recombinant vector comprising a promoter of the present invention, RT-PCR expression pattern of the GUS gene expressed by the promoter of the present invention This is the result of analysis. In this case, EF1α is an elongation factor 1α as a positive control group, Col-0 is a wild type Arabidopsis as a negative control group, and Ad5-1 and Ad5-4 are transformed by inserting the promoter of the present invention. 1 and 4 individuals of the second generation of the Arabidopsis.

The present invention relates to a terrestrial specific promoter and a method of terrestrial specific expression of a target protein using the same. More specifically, the present invention relates to a promoter derived from Arabidopsis can induce the above-ground specific expression of the plant and a method for expressing the target protein in the above-ground specific part of the plant using the same.

Recently, with the development of genetic engineering technology, many researches to improve the traits of plants have been conducted. In particular, much attention has been paid to techniques for obtaining a target useful gene from a transgenic plant, and a promoter involved in gene expression is required when the target useful gene is to be expressed in a transgenic plant.

In general, a promoter is a nucleotide sequence that determines when and how much a gene is expressed, that is, a regulatory region having a function of regulating gene expression. It turned out. Conventionally reported promoters can be classified according to their characteristics and functions, such as a promoter that induces expression at all times and a promoter that induces expression in a time or position-specific manner. Strong promoters. It was initially developed as a promoter used to maximize the expression amount of the gene of interest, representative of the 35S RNA gene promoter of cauliflower mosaic virus (P35S) and the actin gene promoter of rice. These are mainly used for the purpose of antibiotic resistance genes used as selection markers when transforming plants, plant pest suppression genes or the production of substances using plants.

Second, there is a promoter that limits the timing of expression. Many examples have been reported as promoters that limit the expression of the gene of interest only to specific periods of plant development. For example, promoters of genes involved in flowering express the gene only in the flowering period, and promoters of genes expressed by various biological or abiotic stimuli may be included in the timing controlling promoter.

Third, tissue-specific promoters that limit the expression tissue can be cited. These are promoters that are used to accomplish the transformation goal by confining the expression of the gene of interest to specific tissues of the plant. Promoters that induce such tissue specific expression have been reported for each tissue of the plant according to the various purposes to be used.

In particular, among the promoters classified according to functions and features as described above, promoters that induce tissue-specific expression are classified as follows according to tissues expressed in plants. First, systemic expression induction promoters can be cited. As a promoter for inducing expression throughout the whole plant, a promoter of 35S RNA gene of cauliflower mosaic virus (CaMV), which is a promoter used to maximize expression, is used as a representative dicotyledonous plant promoter. As a systemic expression induction promoter for monocotyledonous plants, promoters of rice actin and corn ubiquithin genes are mainly used, and recently, a promoter of rice cytochrome C gene (OsOc1) has been developed (Korea Patent No. 0429335). They are also used to induce the expression of antibiotic or herbicide resistance genes and reporter genes used as selection markers when transforming, as in the promoters that maximize the expression amount. Promoters are the first things to consider when trying to uncover them.

Second, seed specific promoters can be mentioned. A representative example is the rice gluten promoter used to develop golden rice as a promoter of the major storage protein gene of rice, which has been widely used to induce seed-specific expression of monocotyledonous plants. Promoters mainly used to induce seed specific expression include soybean-derived lectin promoters, cabbage-derived napin promoters, and gamma-tocopherol methyl transferase (γ-TMT) in seedlings of Arabidopsis. There are carrot-derived DC-3 promoters and perilla-derived oleosin promoters used in studies to enhance vitamin E production by inducing gene expression.

Third, as a root-specific expression promoter, which has not been commercialized yet, Arabidopsis peroxidase (peroxidase, prxEa) was isolated to confirm root-specific expression, and recently, sweet potato-derived maze gene ( ibMADS ) and sugar induction It has been reported that the ADP-glucose pyrophosphatase (AGPase) gene is isolated to induce the expression of the promoter in the roots and to the transient expression of the roots in carrots and radishes. .

Fourth, other tissue-specific promoters such as leaves, and the related promoters include rice and corn-derived albics (rbcS) ribulose bisphosphate carboxylase / oxygenase small subunit that induces strong gene expression only in photosynthetic tissues such as leaves. Promoter), tomato-derived fruit maturation-specific expression-induced phytoene synthase (PDS) promoter, and oleosin (oleosin) promoter from pollen-specific cabbage of Chinese cabbage flowers.

These seed, root or leaf specific expression promoters are expected to be used for food, food, or major crops used as raw materials of food for agricultural transformation, useful protein accumulation, and useful material generation.

However, the known promoters, in particular, promoters that induce expression in whole tissues of plants, have a disadvantage in that the amount of expression of the target gene is small in other tissues except the leaf and the active cell division of the plants, and also toxic by the promoters. Due to the effect, there is a problem that the transformed plant is dwarfed or not regenerated. In addition, conventional promoters known to induce tissue-specific expression in plants have an advantage of expressing a target gene in a tissue-specific manner, but the amount of expression thereof is insignificant. Therefore, there is an urgent need to discover useful new promoters capable of expressing the genes of interest in tissues and expressing them in large quantities.

Therefore, the present inventors, while studying to find a new promoter capable of tissue-specific expression of the target gene in the plant, the PAtAD5 promoter isolated from Arabidopsis has the activity of inducing the expression of the target gene only in the ground of the plant The present invention has been completed by elucidating.

It is therefore an object of the present invention to provide a ground specific promoter of a plant.

Another object of the present invention is to provide a recombinant expression vector comprising a plant-specific promoter of a plant and a base sequence encoding a protein of interest linked to the promoter.

Another object of the present invention is to provide bacteria and plants transformed with the recombinant expression vector.

In addition, another object of the present invention is to provide a method for specific expression of a terrestrial region of a protein of interest comprising the step of introducing the recombinant expression vector into a plant.

Furthermore, another object of the present invention is to provide a method for producing a plant in which the target protein is specifically expressed at the ground.

In order to achieve the above object of the present invention, the present invention provides a plant-specific promoter of the plant.

The present invention also provides a recombinant expression vector comprising a plant specific promoter and a base sequence encoding a target protein operably linked to the promoter.

The present invention also provides a bacterium transformed with the recombinant expression vector.

The present invention also provides a plant transformed with the recombinant expression vector.

In another aspect, the present invention provides a method for the above-described specific expression of the protein of interest comprising the step of introducing the recombinant expression vector into the plant.

Furthermore, the present invention provides a method for producing a plant in which the target protein is specifically expressed at the ground.

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

The present invention has identified a novel terrestrial specific promoter that specifically induces expression of a gene of interest only in the ground of a plant, and provides a method of specifically expressing a target protein in the ground of a plant using the promoter. There is a characteristic.

The present inventors have identified the part of the genome sequence of the At1g53090 gene in the Arabidopsis genome known in the NCBI database to find a new promoter capable of tissue-specific expression of the gene of interest in plants. That is, in one embodiment of the present invention, based on the nucleotide sequence of the At1g53090 gene of the known Arabidopsis genera produced a specific primer for the portion expected to be the promoter region of the At1g53090 gene, genomic DNA extracted from the wild type Arabidopsis By performing PCR using the prepared primers, a DNA fragment expected to be a promoter region of the At1g53090 gene was obtained, and the nucleotide sequence of the DNA was analyzed (see Example 1). As a result, a PCR reaction product of about 1.9 kb was obtained, and the nucleotide sequence of the DNA obtained in the PCR reaction was described as SEQ ID NO: 1, which was named "PAtAD5 promoter".

The At1g53090 gene of the Arabidopsis has a WD-40 repeat motif, a gene similar to spa1 , a phytochrome A inhibitor, and a protein with the WD-40 repeat motif is generally regulated during cell signaling in eukaryotic cells. It is known to be involved in various functions such as transcriptional regulation of related genes up to the process, and is also known to function to control complex combinations of several proteins. In addition, it is a phytochrome A inhibitor gene containing such a WD-40 repeat motif spa1 is known to act as an inhibitory factor in light-dependent morphogenesis in Arabidopsis seedlings (Ishikawa M., Plant J., 46 (5), 736-46, 2006). Therefore, the present inventors could expect that the At1g53090 gene of Arabidopsis daedae may also be involved in tissue-specific expression of genes in plants since the At1g53090 gene has a function of controlling transcription of many genes in cells.

Thus, the present inventors confirmed the expression of the GUS gene in Arabidopsis by fusing the GUS gene to the PAtAD5 promoter of the present invention in order to confirm whether the PAtAD5 promoter of the present invention is tissue-specifically expressed in the plant. (See Example 4). As a result, it was confirmed that the GUS gene is expressed in the whole body except the root of the Arabidopsis, ie, the leaves, stems, flowers and pods of the Arabidopsis. However, the root of the Arabidopsis was confirmed that the GUS gene is not expressed (see Figure 2).

Therefore, the present inventors were able to confirm that the PAtAD5 promoter of the present invention specifically expressed the target gene only in the ground part of the plant from the above results, and confirmed it again through RT-PCR analysis. That is, in one embodiment of the present invention, after extracting the entire RNA from the leaves, roots, stems and flowers of the Arabidopsis transformed by fusing the GUS gene to the PAtAD5 promoter of the present invention, the RNA as a template and specificity of the GUS gene The expression of the GUS gene in each tissue of the plant was analyzed by performing RT-PCR with the red primer. At this time, the Arabidopsis transformed by fusion of the GUS gene to the P35S promoter known as a promoter for inducing systemic expression was used as a positive control group (see Example 5). As a result, it was confirmed that the GUS gene was expressed in the leaves, stems, and flowers of the transgenic Arabidopsis in which the PAtAD5 promoter of the present invention was inserted, but it was confirmed that the GUS gene was not expressed in the root. On the other hand, in the transgenic Arabidopsis in which the P35S promoter, a systemic expression promoter, was inserted, it was confirmed that the GUS gene was expressed in all tissues including the root (see FIG. 3).

Therefore, it can be seen from the above results that the PAtAD5 promoter of the present invention is a terrestrial specific expression promoter which induces the expression of the target gene in the ground except the root of the plant, and in particular, induces the expression of the gene in the leaves, stems and flowers of the plant. It was found that the promoter.

Therefore, the present invention provides a plant-specific promoter of the plant part comprising the nucleotide sequence of SEQ ID NO: 1.

In the present invention, the ground part of the plant refers to all parts of the plant except for the root of the plant, and preferably, the leaves, stems, pods and flowers of the plant. Therefore, since the promoter of the present invention induces specific expression of the target gene only in the ground part of the plant, the promoter of the present invention can produce a recombinant expression vector that specifically expresses the target protein derived from the outside only in the ground part of the plant. .

Accordingly, the present invention provides a recombinant expression vector comprising a plant-specific promoter and a base sequence encoding a target protein operably linked to the promoter.

The "promoter" is a gene region generally located upstream of the coding region including the point at which transcription is initiated, and often affects gene expression in response to TATA boxes, CAAT box regions, and external stimuli that regulate gene expression. It consists of an enhancer that promotes the expression of almost all genes regardless of site, position and orientation. In addition, among these promoters are promoters that induce expression in all tissues and promoters that induce expression in a time or position-specific manner, and in particular, the promoter according to the present invention induces expression only in the ground part of the plant. .

The term "expression vector" refers to a plasmid, virus or other medium known in the art into which a promoter of the present invention and a base sequence encoding a target protein operably linked to the promoter can be inserted or introduced. . The above-ground expression promoter of a plant according to the present invention and a base sequence encoding a target protein operably linked to the promoter may be operably linked to an expression control sequence, wherein the operably linked gene sequence and expression control sequence are It can be included in one expression vector that contains the selection marker and replication origin together. “Operably linked” can be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. An "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 a promoter of the present invention using a conventional vector used for protein expression as a base skeleton and inserting a base sequence encoding a target protein downstream of the promoter. have. Thus, vectors that can be used in the present invention include 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. And plasmids such as Ti plasmid, plant viral vectors, and binary vectors such as pCHF3, pPZP, pGA, and pCAMBIA series. 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.

The target protein that can be used in the present invention may be any target protein of external origin, ie, an exogenous protein or an endogenous protein and a reporter protein, which is desired to be specifically expressed above the plant by the promoter of the present invention. 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.

Therefore, if the promoter of the present invention is used in the case of crops ingesting the upper part of the plant, for example, leaves or stems, the target protein can be ingested together with the leaf or stem by expressing the useful target protein in the upper part of the plant. You can develop crops.

In one embodiment of the present invention is a green fluorescent protein gene Egfp and blue color gene of the Gus is operably linked to PAtAD5 promoter of the present invention having the base sequence of SEQ ID NO: 1 to known pBGWFS7 vector that has the form of a reporter system that is connected is inherent in the upstream section of Egfp and Gus gene recombination An expression vector, pBGWFS7-PAtAD5, was prepared (see Example 2).

A recombinant expression vector comprising a promoter of the present invention and a nucleotide sequence encoding a protein of interest linked to the promoter can be introduced into a host cell using methods known in the art. The method for introducing the recombinant expression vector according to the present invention into a host cell may include, but is not limited to, calcium chloride (CaCl 2) and heat shock, particle gun bombardment, and silicon carbide whiskers. (Silicon carbide whiskers), sonication (sonication), electroporation (electroporation) and precipitation by PEG (Polyethylenglycol) may be used.

Accordingly, the present invention provides a host cell transformed with a recombinant expression vector comprising a promoter of the present invention and a nucleotide sequence encoding a target protein operably linked to the promoter. The host cell is preferably a bacterium, and in one embodiment of the present invention, Agrobacterium tumefaciens GV3101 is used.

In addition, the recombinant expression vector according to the present invention can be introduced into plants using methods known in the art. For example, but are not limited to Agrobacterium (Agrobacterium sp.) - method according to the parameters, the particle gun bombardment method (particle gun bombardment), silicon carbide whisker methods (Silicon carbide whiskers), ultrasound treatment (sonication), electrical Precipitation by electroporation and polyethylene glycol (PEG) may be used. Preferably Agrobacterium sp . ) -Mediated methods can be used.

Accordingly, the present invention provides a transformed plant by introducing into the plant a recombinant expression vector comprising a promoter of the present invention and a nucleotide sequence encoding a target protein operably linked to the promoter.

The transformed plant according to the present invention can be obtained through the sexual breeding method or asexual breeding method which is a conventional method in the art. More specifically, the plant of the present invention may be obtained through oily breeding, which is a process of producing seeds from the pollination process of flowers and breeding from the seeds. In addition, after transforming a plant with a recombinant expression vector according to the present invention it can be obtained through the asexual propagation method which is a process of induction, rooting and soil purification of the callus according to a conventional method. That is, the explants of the plant transformed with the recombinant expression vector of the present invention are inoculated in a suitable medium known in the art, and then cultured under appropriate conditions to induce the formation of callus. do. After about 2 weeks, the shoots are transferred to rooting medium to induce roots. The transformed plant according to the present invention can be obtained by inducing roots and then transplanting them into the soil to purify them. In the present invention, the transformed plant may include not only the whole plant, but also tissues, cells, or seeds obtainable therefrom.

In addition, the present invention provides a method for terrestrial specific expression of a target protein comprising introducing into a plant a recombinant expression vector comprising a promoter of the present invention and a nucleotide sequence encoding a target protein operably linked to the promoter. to provide.

Furthermore, the present invention provides a method for producing a plant in which the target protein is specifically expressed in the ground part.

In the present invention, the plant may be a dicotyledonous plant or a monocotyledonous plant, and the dicotyledonous plant is not limited thereto, but the Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, Chinese cabbage, radish, cabbage, lettuce, peach , Pears, strawberries, watermelons, melons, cucumbers, carrots, celery and rapeseed, and the monocotyledonous plants may include, but are not limited to, rice, barley, wheat, rye, corn, sugar cane, oats and onions.

Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not necessarily limited to these Examples.

<Example 1>

Identification of promoter region of At1g53090 gene from Arabidopsis

In order to secure a terrestrial specific promoter, the inventors prepared specific primers for the part expected to be a promoter part of the gene, based on the nucleotide sequence of At1g53090, a genomic gene of Arabidopsis. Then, in order to extract genomic DNA from wild-type Arabidopsis rosette leaves, a small amount of Arabidopsis leaf tissue was placed in an Eppendorf tube and isolated and purified using a genomic DNA extraction kit (SolGent Co., Ltd.). The DNA eluate was quantified by measuring A ₂₆₀ / A ₂₈₀ using an ultraviolet absorbance analyzer (UV spectrophotometer, Beckman Coulter DU650). Then, PCR was performed using the extracted genomic DNA as a template and primers prepared above, that is, primers of SEQ ID NO: 2 and SEQ ID NO: 3 described below. At this time, the PCR conditions were denatured at 95 ° C for 10 minutes, followed by 30 cycles of 1 minute at 94 ° C, 1 minute at 55 ° C and 2 minutes at 68 ° C for 1 cycle, followed by 10 minutes at 68 ° C. Subsequently, DNA sequences obtained from the PCR reaction were analyzed (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, second edition, 1989).

SEQ ID NO: 2: AD5 B1 forward primer

5'-AAAAAGCAGGCTTGGCAATCCTAATAACCATC-3 ’

SEQ ID NO: 3: AD5 B2 reverse primer

5'-AGAAAGCTGGGTTCCTCTGGATTCTGCAATAC-3 ’

As a result, a PCR reaction product corresponding to about 1.9 kb expected to be a terrestrial expression promoter was obtained, and the nucleotide sequence of the DNA product obtained in the PCR reaction was represented by SEQ ID NO: 1, which was named "PAtAD5 promoter". It was.

<Example 2>

Preparation of Recombinant Expression Vector Comprising PAtAD5 Promoter

In order to transform the PAtAD5 promoter isolated in Example 1 into Arabidopsis, a recombinant expression vector including the PAtAD5 promoter was prepared. To this end, first, a production vector in which a PAtAD5 promoter was inserted into pBGWFS7, a gateway vector purchased from the University of Ghent, Belgium, was prepared. That is, the PCR product obtained in Example 1 is a primer primer B1 (SEQ ID NO: 4) and primer B2 (SEQ ID NO: 5) which is a primer including the entire bacterial side specific recombinant nucleic acid sequence site when bacteria are infected by bacteriophage PCR was performed again. At this time, the PCR conditions were denatured at 95 ° C. for 3 minutes, followed by 5 cycles of reaction for 1 minute at 94 ° C. for 30 seconds, 45 ° C. for 30 seconds, and 68 ° C. for 2 minutes, and then at 94 ° C. for 30 seconds and 55 ° C. 20 cycles were repeated, and the reaction was repeated for 1 minute at 30 seconds and 68 ° C. for 2 minutes, and the reaction was performed at 68 ° C. for 10 minutes. When the bacteria were infected by the bacteriophage, the PCR products were subjected to a BP recombination reaction with a pDONR221 vector (Invitrogen Biotech, USA) containing a bacteriophage-specific recombinant nucleic acid sequence site, that is, a BP reaction buffer, 200ng of the PCR product, pDONR221 After mixing the 150ng vector and the gateway BP clonease enzyme mixture well and reacting at 25 ° C. overnight, pDONR-PAtAD5, an intermediate cloning vector into which the PAtAD5 promoter was inserted, was prepared. Then, the prepared pDONR-PAtAD5 vector is Egfp, which is a green fluorescent protein gene, to finally produce a plant transformation recombinant expression vector including a PAtAD5 promoter. And LR recombination reaction, ie, LR reaction buffer, 300ng of pDONR-PAtAD5 vector, and gateway LR clonease enzyme mixture, which are well mixed with the pBGWFS7 vector incorporating the reporter system in which the blue chromogenic gene Gus is linked. The reaction was carried out overnight at 25 ° C. to produce a recombinant expression vector pBGWFS7-PAtAD5. At this time, the pBGWFS7 vector has a spectinomycin resistance gene and includes a Bar gene resistant to herbicide, a plant selection factor. A map of pBGWFS7-PAtAD5, a recombinant expression vector comprising the PAtAD5 promoter region of the present invention prepared by the above method, is shown in FIG. 1.

SEQ ID NO 4: Adapter Primer B1

5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3 '

SEQ ID NO 5: Adapter Primer B2

5'-GGGGACCACTTTGTACAAGAAAGCTGGGT-3 '

<Example 3>

Preparation of Arabidopsis Transformed with Recombinant Expression Vector Comprising PAtAD5 Promoter

PBGWFS7-PAtAD5, the recombinant expression vector prepared in Example 2, was introduced into the Agrobacterium tumefaciens GV3101 strain according to the transformation method using liquid nitrogen, and then using the wild type Arabidopsis col-o ( Arabidopsis). thaliana ecotype Col-o) was transformed. To this end, Agrobacterium tumefaciens GV3101 strain transformed with the pBGWFS7-PAtAD5 vector was incubated for 48 hours in a 5 ml LB medium containing antibiotics, and then the culture was centrifuged at 4000 rpm for 10 minutes. Thereafter, suspended cells were suspended in a solution containing 5% sucrose to prepare an Agrobacterium culture and 250 ppm tween 20 was added. The seedlings to be transformed are grown for 4 weeks in a 22 ° C. incubator for 16 hours during the day and 8 hours at night after seeding, and then at least 3-4 secondary rods are induced by removing the primary rods from plants. The Agrobacterium culture solution prepared above was sprayed onto the buds to transform the Arabidopsis. Thereafter, overnight incubation was carried out with sufficient humidity and dark conditions, followed by incubation for 5 more days in a 22 ° C. incubator for 16 hours and night for 8 hours, and the transformation was repeated in the same manner as above. Seeds were harvested from the transformed Arabidopsis cultivars after growing for about 3-5 weeks in 16 hours day and 8 hours night conditions in a 22 ° C. incubator until the tissue (silique) had fully matured to brown. The harvested seeds were sown again and the transformants of the T1 generation, which survived, were sprayed with 0.3% herbicide, Basta, on the first day of the 10th day and the second day of the 17th day. The T1 transformants were further grown for about two months in a 22 ° C. incubator for 16 hours day and 8 hours at night until the pod tissue (silique) was fully matured, and then harvested T2 seeds.

<Example 4>

Investigation of GUS Expression by PAtAD5 Promoter in Transgenic Arabidopsis

In order to confirm whether the PAtAD5 promoter of the present invention induces tissue expression in a plant, the expression of the GUS gene in each tissue of the Arabidopsis transformed with a recombinant expression vector into which the PAtAD5 promoter of the present invention is inserted The aspect was analyzed. That is, some of the T2 seeds harvested from the Arabidopsis transformed in Example 3 germinated in water to observe the expression pattern of the GUS (blue color gene) gene in seedlings on the 7th day, and some of them were seeded and grown on top soil. The expression of the GUS gene was observed at various stages of growth from various tissues such as leaves, stems, roots, flowers and pods of transgenic Arabidopsis plants, which were sprayed with two 0.3% bastards on day 10 and day 17. The control group was used to germinate non-transformed Arabidopsis. Observation of the expression of the GUS gene was carried out in each of the germinated seedlings and the tissues of the transformed Arabidopsis, GUS- assay buffer (X-gluc (cyslohexyl ammonium salt) 20mM, NaH ₂ PO ₄ H ₂ O 100mM, NaEDTA 10mM) , Triton-X-100 0.1%, pH7.5) and treated overnight at 37 ℃, discarded the buffer solution, washed with water and then immersed in 70% ethanol once again at 37 ℃ chlorophyll (chlorophyll once every hour ) Until it is completely removed. The plant was then observed under an anatomical microscope (Olympus, JP / GX41).

As a result, it was confirmed that the GUS gene was expressed by the PAtAD5 promoter of the present invention in the whole seedlings except the roots in the seedlings germinated in water at 7 days. In contrast, no GUS gene expression was observed in the control transgenic Arabidopsis. In addition, it was confirmed that GUS gene was expressed in various tissues of transgenic Arabidopsis cultivars, such as leaves, stems, flowers, and pods, but was rarely expressed in roots. No expression of the GUS gene was observed in the leaves, flowers and pods of the transgenic Arabidopsis (see FIG. 2).

Therefore, it was confirmed from the above results that the PAtAD5 promoter of the present invention is a promoter that specifically induces gene expression in above-ground parts of plants except roots, that is, leaves, stems, pods and flowers of plants.

<Example 5>

Analysis of Plant Surface Expression of PAtAD5 Promoter by RT-PCR Analysis

In Example 4, the inventors of the present invention confirmed that the PAtAD5 promoter of the present invention specifically induced gene expression only in the ground part of the plant, and performed the RT-PCR method to confirm this again. To this end, the total RNA was isolated from the leaves, roots, stems and flowers of Arabidopsis transformed with a recombinant expression vector comprising the PAtAD5 promoter of the present invention obtained by cultivating the T2 generation seed obtained in Example 3. At this time, the non-transformed Arabidopsis Columbia-0 was used as a negative control group, and the Arabidopsis transformed with a recombinant expression vector including a P35S promoter known as a systemic expression promoter was used as a positive control group. Isolation of the RNA, first, 1 g of each tissue of each plant was frozen with liquid nitrogen, crushed with a mortar and transferred to a 2 ml tube, and then 500 μl of RNA extraction buffer (50 mM sodium acetate pH 5.5, 150 mM LiCl) was added to each tube. , 5 mM EDTA, 0.5% SDS) and 500 μl phenol were added and mixed. After the mixture was treated at 65 ° C. for 10 minutes and mixed at room temperature for 15 minutes using a stirrer (rotary shaker), centrifuged at 4 ° C. for 10 minutes at 10000 rpm, and then the supernatant was carefully transferred to a new tube. 500 µl of chloroform was added thereto, mixed well, and centrifuged again to obtain a supernatant. 0.6M volume of 8M lithium chloride (LiCl) was added to the obtained supernatant and then stored at -20 ° C for at least 2 hours. Thereafter, centrifugation at 12,000 rpm for 20 minutes at 4 ℃ to wash the RNA precipitate with 4M LiCl and 80% ethanol one by one to dissolve the final RNA precipitate in 50μl of distilled water. The RNA dissolved in the distilled water was quantified by measuring A260 / A280 using an ultraviolet absorbance analyzer (UV spectrophotometer, Beckman Coulter DU650). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using 50 μM of ThermoScript RT-PCR system (Invitrogen Biotech, USA) as a template with approximately 1 μg of each extracted RNA as a template. oligo dT primer and reaction buffer (10X RT buffer, 25mM MgCl ₂ , 0.1M DTT, RNase OUT, SuperScript _TM RT) were added for 50 min at 50 ° C, 5 min at 85 ° C, and then cooled on ice. 1 μl of H was added and treated at 37 ° C. for 20 minutes to synthesize cDNA. Subsequently, EF-1α as an extension of Arabidopsis as a control for quantitative analysis and primers of SEQ ID NO: 6 and SEQ ID NO. PCR reactions were performed using primers of SEQ ID NO: 8 and SEQ ID NO: 9 (elongation factor-1α) primers. The PCR reaction was denatured at 94 ° C. for 5 minutes, followed by 25 cycles of reaction for 30 seconds at 94 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C., followed by 25 cycles of reaction at 72 ° C., followed by agarose gel. Electrophoresis on the PCR product was confirmed. The PCR reaction results are shown in FIG. 3.

SEQ ID NO: 6 GUS forward primer

5'-ACCTGCGTCAATGTAATGTTCTGC-3 '

SEQ ID NO: 7: GUS reverse primer

5'-CTCCCTGCTGCGGTTTTTCA-3 '

SEQ ID NO: EF-1α forward primer

5'-GTTTCACATTAACATTGTGGTCATT-3 '

SEQ ID NO: EF-1α reverse primer

5'-GAGGTACCAGTAATCATGTTCTTG-3 '

As a result, as shown in Figure 3, it was confirmed that the GUS gene was expressed in the leaf, stem and flower tissue of Arabidopsis transformed with the recombinant expression vector containing the PAtAD5 promoter of the present invention, but the expression of the GUS gene in the root The product could not be identified. On the other hand, in the transgenic Arabidopsis inserted with the P35S, a systemic expression promoter used as a positive control, it was confirmed that the GUS gene was expressed in all tissues including the roots.

Therefore, as a result of the above, it can be seen that the PAtAD5 promoter of the present invention is a terrestrial specific promoter that induces the expression of genes in the ground except for the root of the plant, and the PAtAD5 promoter of the present invention is particularly a leaf, stem and flower tissue of the plant It can be seen that it is a promoter that induces the expression of genes.

As described above, the above-ground specific promoter of the present invention has an effect of excellent tissue specificity capable of specifically inducing the expression of a gene of interest only in the above-ground part of the plant except the root of the plant. Therefore, the above-ground specific promoter of the present invention can be usefully used in the development of a transgenic plant that can produce a large amount by expressing a high value-added useful component in the above-ground plant.

<110> Rural development administration <120> Above-Ground Specific Promoter and Above-Ground Specific          Expression Method of Target Protein Using the Same <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 1939 <212> DNA <213> Arabidopsis thaliana <400> 1 tggcaatcct aataaccatc tctttgcggt cgaattcgat gttttccaag acaaacgttt 60 tggtgatatt aatgataatc atgttggtgt taatattaac tctgttaact caaaggtttc 120 tgagaaagct gggtattgga ttcagacaag aactagaggg aagaatcaat ggttgtttaa 180 ggaagtgaag cttagtagcg gagacaacta caaagcttgg attgagtata agaacagtaa 240 agtcattgtt tggcttgcac ctgcgcattt aaagaaaccg aagaggccat tgatcgaaac 300 acaagttgat ctctcggagg tggttcttga gactatgtat accggctttt ccggttcgat 360 gggtcgtggc gtcgagcgtc acgacatttg gagctggagt tttgagaaca ccgccaaaaa 420 caattagttc tgattttggt cttgtttggt ttgatttatg tttgttaccg gtttaacaat 480 tatgtatttt ctcttgattt ttattcaaaa gtggttaaaa agagagtacg tataaaatgt 540 caaaatcaaa tttaaaatta ctttgatcga ttaaaccatt aaaaaaaatc ctgagaagaa 600 aacatgcaca ctgtcccgga ccatcaccgt caatgatctt cttggacatg catcattgat 660 tttgtataaa taaagactcg ttaaaagcca aagcacaacc acgtggtatt gtggtcatcg 720 tcctcatcaa attgaatcag tttgttcatg tcacaagttt cttaacataa gtttttaaaa 780 gatattacta aaatttaaaa ctaaaatatt gacaaatcca cgatatataa ggttaaagct 840 atcacagttt ctaggtcgta tcattttcta caaaaatctt gcattacatt tcagatggtt 900 tggtttcaat aaaaaaacag agtttttttt tttttttcat tgataaattt gtgtatgtaa 960 tctaatcata gaaaaagaaa aaacacgtgg cgtgtccgga cgcatgtgat tttgtaattt 1020 accacttacg ttgtcgactt gtctcaatcc attccatgga aatggaaact cccctctggt 1080 tattaaatgc tcccaccaat acattctctt tcctttcacc aaaaccaata atacaaacaa 1140 ttttgttttt ctcattcaag attttcaaag tacaatcatt tctttaggta ctttctcttg 1200 taataaggct tcaaatatgt tattcaaaaa ttattttggt cattttaaat tcctattata 1260 gccttacaaa aatgtgttat attatttgga ccacaacata atttaaacat tcctcttgaa 1320 gaaaattggt cacttcaaaa aagaacaaaa ttcactttga cctcatggtt tacttatatt 1380 tacatatatg gcccaaaacc aaaatacgaa aaaggtttta gctttacgtt cttttgattc 1440 ctaggtcgtc gtttaaccac gttgcttgcg tggaaagaga agagaagtga agattatcaa 1500 aaagcgaaaa ataaaaccct cactgtgaag taaaaatcgt aggcccaaaa tagatttctc 1560 ttagccgaga atttaatttt tatgaatctg attattgctt ttctacgaac tcagtttttg 1620 cgatgtgtgg ttctcaaaaa aggatcgttt cttcggtgcc tcttagtctc tcaagtactt 1680 cttcttcttc ttatcttctt cttcgctttt cttttatata tagagagagt ttagtggtca 1740 agaagcttcc tcgtgtttgt ctctttatgt aatcaaaggt tgcattttta ttcgaatata 1800 ttttctggtt ttggtgtttt ctgtcaaagg gtcttttaaa aatcttaact ttcgttctaa 1860 atttctttac tttatttcga ttctaactta gggcttttgg gctgggatca tgaaatgaag 1920 tattgcagaa tccagagga 1939 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> AD5 B1 froward primer <400> 2 aaaaagcagg cttggcaatc ctaataacca tc 32 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> AD5 B2 reverse primer <400> 3 agaaagctgg gttcctctgg attctgcaat ac 32 <210> 4 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter B1 primer <400> 4 ggggacaagt ttgtacaaaa aagcaggct 29 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> adapter B2 primer <400> 5 ggggaccact ttgtacaaga aagctgggt 29 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GUS forward primer <400> 6 acctgcgtca atgtaatgtt ctgc 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUS reverse primer <400> 7 ctccctgctg cggtttttca 20 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> EF-1a forward primer <400> 8 gtttcacatt aacattgtgg tcatt 25 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> EF-1a reverse primer <400> 9 gaggtaccag taatcatgtt cttg 24  

Claims (14)

Terrestrial specific promoters of plants comprising the nucleotide sequence of SEQ ID NO: 1. The promoter of claim 1, wherein the ground portion is selected from the group consisting of leaves, stems, pods, and flowers. The recombinant expression vector comprising the promoter of claim 1 and a base sequence encoding a protein of interest operably linked with the promoter. The recombinant expression vector according to claim 3, wherein the recombinant expression vector is pBGWFS7-PAtAD5 described in FIG. A bacterium transformed with the recombinant expression vector of claim 3. A plant transformed with the recombinant expression vector of claim 3. A method of expressing a terrestrial portion of a protein of interest comprising introducing the recombinant expression vector of claim 3 into a plant. A method for producing a plant in which the target protein is specifically expressed on the ground, comprising introducing the recombinant expression vector of claim 3 into a plant. 8. The method of claim 7, wherein the plant is a dicotyledonous or monocotyledonous plant. The method of claim 9, wherein the dicotyledonous plants are Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot, celery and rapeseed Method characterized in that it is selected from the group consisting of. The method of claim 9, wherein the monocotyledonous plant is selected from the group consisting of rice, barley, wheat, rye, corn, sugar cane, oats and onions. The method of claim 8, wherein the plant is a dicotyledonous plant or monocotyledonous plant. The method of claim 12, wherein the dicotyledonous plants are Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot, celery and rapeseed Method characterized in that it is selected from the group consisting of. The method of claim 12, wherein the monocotyledonous plant is selected from the group consisting of rice, barley, wheat, rye, corn, sugar cane, oats and onions.

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