KR101822708B1 - A xylem-specific promoter and uses thereof - Google Patents

Abstract

The present invention relates to a plant cell-specific expression cassette of a target gene, a recombinant expression vector comprising the expression cassette, and a gene expression vector comprising the expression vector, wherein the expression vector comprises a plant cell specific expression cassette comprising the promoter and the target gene operably linked thereto, Bacteria, and plants transformed with the above expression vectors. The present invention also relates to a method for producing a transgenic plant expressing a gene of interest specifically for a woody plant using the recombinant expression vector, and a method for producing a target protein by expressing a gene of interest in the transgenic plant. The present invention also relates to a cassette for introducing a target gene for producing a plant-specific gene expression cassette of a target gene, which comprises the promoter and a gateway cassette, a vector for introducing a target gene comprising the introduction cassette, The present invention relates to a method for producing a wood-specific expression vector of a desired gene by using a vector for expression of the gene of interest.
The plant-specific promoter of the present invention can induce superior wood-tissue-specificity and strong expression, independent of the developmental stage of the plant and external and internal environmental stresses.

Description

[0001] The present invention relates to wood-specific promoters and their use,

The present invention relates to a plant cell-specific expression cassette of a target gene, a recombinant expression vector comprising the expression cassette, and a gene expression vector comprising the expression vector, wherein the expression vector comprises a plant cell specific expression cassette comprising the promoter and the target gene operably linked thereto, Bacteria, and plants transformed with said expression vectors.

The present invention also relates to a method for producing a transgenic plant expressing a gene of interest specifically in woody parts using the recombinant expression vector, and a method for producing a target protein by expressing a gene of interest in the transgenic plant.

The present invention also relates to a cassette for introducing a target gene for producing a plant-specific gene expression cassette of a target gene, which comprises the promoter and a gateway cassette, a vector for introducing a target gene comprising the introduction cassette, The present invention relates to a method for producing a plant-specific gene expression vector of a desired gene by using a vector for expression of a target gene.

Unlike herbaceous plants, woody plants are formed by the development of vascular cambium, forming secondary xylem, wood and mature xylem. The woody part serves as a mechanical support not only for the transport of the necessary water and nutrients but also for the large ground part of the woody plant to stand upright against gravity. The woody part consists of a vessel element, a xylary fiber cell, and a xylem parenchyma. Among these, the conduit element and ductal fibroblast are differentiated into PCD (Programmed Cell Death) ) Through which the cells die and take on a structural role (water transport, support role). Conduit flow cells serve as living cells and provide necessary materials inside the woody part.

On the other hand, the technology of transgenic plant development using biotechnology is very important because it can newly introduce useful traits in crops, and the expression pattern of genes determining useful traits is controlled by specific promoters. The DNA region located upstream of the transcription initiation point of the gene and determining the expression level and pattern such as the transcription initiation and frequency of the gene is generally referred to as a promoter and transcription initiation by the promoter is referred to as RNA The specific transcription factors that activate the polymerase, the RNA polymerase, and the basal transcription complex, together, bind to the promoter nucleotide sequence.

The 35S promoter derived from Cauliflower mosaic virus is a promoter that constantly expresses genes in all tissues of plants and is most frequently used in plant transformation. However, it is well known that when this 35S promoter is used to express an exogenous useful gene constantly in all tissues, it can often cause abnormal growth of the plant. Therefore, there is a desperate need for a technology that can regulate expression of an exogenous useful gene according to purpose, that is, a gene capable of expressing a gene in tissue, developmental stage, or environment-specific manner.

If we can control exogenous gene expression specifically for woody parts, breakthrough biomass improvement can be achieved in three aspects: Firstly, it is possible to quantitatively and qualitatively improve the woody part through the expression of biosynthetic genes of secondary cell wall constituents (cellulosic, lignin, and xylan). Secondly, the resistance of the woody part to insect pests is increased through the expression of antibacterial / antibacterial proteins. The color and function of the woody part can be improved by the expression of an antioxidant-forming gene such as anthocyanin, tannin, flavonoid, carotinoid, etc. (Korean Patent Publication No. 10-2006-0013380; Plant Biotechnology Journal, 2012, 10, 587- 596).

Accordingly, the present inventors have made intensive efforts to develop an expression system capable of specifically expressing in the woody part. As a result, the present inventors have identified a promoter of PtrMX3 gene that is specifically expressed in woody part in poplar, The present inventors have completed the present invention by confirming the expression of gene specific to woody plants in transgenic plants. Accordingly, the desired gene can be expressed specifically in the woody part of the plant by using the expression vector containing the PtrMX3 promoter of the present invention. Therefore, the present invention can be widely used .

One object of the present invention is to provide a wood-specific promoter of a plant.

Another object of the present invention is to provide a plant-specific gene expression cassette of a target gene, which comprises the promoter and a target gene operably linked thereto.

It is still another object of the present invention to provide a cassette for introducing a target gene for producing a plant-specific gene expression cassette of a target gene, which comprises the promoter and the gateway cassette.

It is another object of the present invention to provide a recombinant expression vector comprising said expression cassette.

It is still another object of the present invention to provide a vector for introducing a target gene comprising the introducing cassette.

It is still another object of the present invention to provide a bacterium containing the recombinant expression vector.

It is another object of the present invention to provide a plant transformed with said recombinant expression vector.

It is still another object of the present invention to provide a method for producing a plant-specific gene expression vector of a target gene using the vector for introducing the desired gene.

It is still another object of the present invention to provide a method for producing a transgenic plant expressing a gene of interest, specifically a wood-based plant, using the recombinant expression vector.

It is still another object of the present invention to provide a method for producing a target protein by expressing a target gene in the transgenic plant.

In order to achieve the above object, the present invention provides, as one embodiment, a wood-specific promoter of a plant, which comprises the nucleotide sequence of SEQ ID NO: 3.

In the present invention, the term "woody part" refers to a passage of water and nutrients absorbed from the root, which is also referred to as a manganese or throat, and most of the cells constituting it are lignified to form a thick wall. In the case of trees, most of the coarse stems are occupied by the bulb, so the wood is solid. The woody part is a complex structure composed of several kinds of tissues, and the composition thereof is different depending on the kind or part of the plant. In the elements constituting the woody part, there are a water tube composed of long cells in the same direction as the axis of the engine, a vasculature, a vesicular fiber, a vesicular vesicle and a radial tissue having an axis in the radial direction of the vesicle.

Among them, the water channel serves as a channel for water, while for water plants, gymnosperms and ferns is a vase. However, there are some barn plants in the stem and leaf veins of saphenous plants, and some plants do not have any water tubes like the stratum of Lamiaceae and they serve as a passage for water. On the contrary, there is a water pipe such as a horse plant of a gymnosperm plant or a horse fern which is a fern. Among the gymnosperms, there is a vase in the radiation structure. Mulberry fiber is a type of mechanical structure with a solid fiber structure, and is often found in the tailings of a tree. Because of this, the stem can serve to mechanically support the plant. Cells that form the fiber of the metamorphose fiber are dead cells with a thick membrane, with both ends of a cylindrical shape with a pointed circular cross section with a circular or polygonal shape. Gypsum plants and ferns do not have fiber bundles. The milk tubular tissues are also storage tissues such as starch and fat.

In the present invention, the "woody part" may include at least one of a water pipe, a tub, a pipe fiber, a water pipe or a radiation pipe.

In the present invention, the term " woody part specific "means that the gene expression level is higher in woody parts than in other tissues, the expression level is low in other tissues but relatively high in woody part, In other tissues, the gene may not be expressed but expressed only in the woody part.

The term "promoter " in the present invention is a gene region located upstream of an encoding site, which generally includes a point at which transcription is initiated, and is usually a TATA box for regulating gene expression, a CAAT box site, And an enhancer that promotes almost all gene expression regardless of the location and direction of the gene in response to the gene expression. In addition, among these promoters, there are a promoter that induces expression constantly in all tissues and a promoter that induces expression in a time or position specific manner. Specifically, the promoter according to the present invention is a PtrMX3 promoter that is expressed specifically in woody plants in plants .

The PtrMX3 promoter is a promoter identified by the present inventors in Poplar, and is capable of significantly increasing the expression of a target gene that is specific to a woody tissue and desired to be specifically expressed in a woody part. Specifically, the PtrMX3 promoter may be a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3, and may contain not only the nucleotide sequence of SEQ ID NO: 3 but also at least 70%, more preferably at least 80% Or more, more particularly 95% or more, and most particularly 98% or more of the nucleotide sequence.

In the present invention, first, a gene specifically expressed in the mossy part of a plant is found, and the promoter of the gene is identified. The recombinant expression vector containing the sequence of the target gene operably linked to the promoter sequence and downstream of the promoter The present inventors completed the invention by confirming that the target gene is expressed specifically in the woody part in the transformed plant transformant.

In a specific example of the present invention, it was confirmed that Potri.013G007900.1 gene (Fig. 5, SEQ ID NO: 1 and SEQ ID NO: 2) derived from poplar was specifically expressed in woody part of poplar (Figs. 1 to 4) The sequence (SEQ ID NO: 3) of the wood-specific promoter (PtrMX3 promoter) located upstream of the gene was identified. The PtrMX3 promoter can specifically express the target gene located downstream in the woody part of the plant.

In another specific embodiment of the present invention, transgenic Arabidopsis thaliana and transgenic poplar were prepared using the PtrMX3 :: GUS expression vector containing the PtrMX3 promoter sequence (SEQ ID NO: 3) and the GUS reporter gene, (Fig. 7 to Fig. 8), and these wood-specific gene expressions were detected at the stage of development of the plant (Fig. 9) or the internal and external stresses of the plant (Figs. 10 and 11). Therefore, the plant-specific gene promoter of the present invention can be usefully used for expressing a desired gene in a woody part of a plant according to purposes.

In another aspect, the present invention provides a plant cell specific expression cassette of a gene of interest, comprising the promoter and a target gene operably linked thereto.

In yet another aspect, the present invention provides a cassette for introducing a target gene for producing a plant-specific gene expression cassette of a target gene comprising the promoter and a gateway cassette.

Expression of gene specific to promoter and woody part of plant is as described above.

The term "operably linked" in the context of the present invention is intended to encompass transcription and any translation regulation of the coding sequence (s) into the sequence (s) that allows the regulatory elements to direct expression of the encoding sequences by the action of the host cell Quot; refers to any structure in which the elements are attached to the encoding sequence.

As used herein, the term "cassette " refers to a nucleotide sequence capable of expressing an inserted gene when inserted such that the particular gene is operably linked to one or more regulatory sequences present in the nucleotide sequence. Thus, an "expression cassette" may comprise the gene of interest, and the expression cassettes of the invention are useful for promoting the expression of the gene of interest in a wood-specific manner. In addition, the cassette of the present invention may include a gateway cassette sequence so that the target gene can be easily introduced. These cassettes represent "cassettes for introduction" according to the present invention.

According to the present invention, the cassette may comprise a PtrMX3 promoter and a gene of interest encoding one or more proteins operably linked thereto. Also, one promoter or the same number of promoters may be operably linked to two or more (i.e., different) genes encoding the protein in the cell.

In the present invention, the term "target gene" refers to a gene that can be introduced to produce a target protein in a cell and encodes the target protein. The target gene or the target protein may be varied depending on the purpose of use in the transgenic plant, and may be any gene or protein that can be expressed in the plant. That is, since the feature of the present invention is the woody part-specific expression promoter of the plant, the target gene or the target protein can be appropriately selected by those skilled in the art. The target protein is expressed to a greater extent than the reference cell (wild-type cell), and can be specifically expressed in the woody part.

The gateway expression system is composed of an entry vector and a destination vector. Specifically, the entry vector is a vector having attL1 and attL2 at both ends of the target gene, and the destination vector is a vector containing attR1 and attR2. The entry vector and the target vector are subjected to LR reaction by the recombinase, and attR1 and attR2 of the target vector are replaced with attL1 and attL2 of the entry vector, respectively. In this process, the target gene contained in the entry vector is transmitted to the destination vector.

In the present invention, the "gateway cassette" is a cassette capable of producing a vector for introducing a target gene, and may be produced or marketed. Particularly cassettes comprising attR1, ccdB and attR2 sequences, but are not limited thereto.

The attR1-CmR-ccdB-attR2 cassette, which is commercially available from among the above-mentioned gateway cassettes, is a recombinant 25-bp sequence (attR1, attR2), a chloramphenicol resistance gene (CmR), a cleavage gene Which acts as a toxin, so it can be amplified using special bacteria that do not die by ccdB and, if not needed, to kill bacteria with ccdB as normal bacteria.

In a specific embodiment of the present invention, as shown in FIG. 6, a kanamycin or basta resistance gene, a PtrMX3 promoter, an EGFP and a GUS reporter gene are used to confirm the wood-specific gene expression of the PtrMX3 promoter Expression cassettes were prepared. As shown in FIG. 12, a cassette for introducing a target gene containing a kanamycin or Basta resistance gene, a PtrMX3 promoter and a gateway cassette was prepared so that the target gene could be easily introduced.

In yet another embodiment, the invention provides a recombinant expression vector comprising said expression cassette.

In yet another embodiment, the present invention provides a vector for introducing a target gene comprising the introducing cassette.

The expression cassette and the introduction cassette are as described above.

The recombinant expression vector is a vector capable of producing a protein encoded by a target gene introduced into a plant including the expression cassette. Specifically, the recombinant expression vector is a vector capable of producing a protein encoded by the target gene specifically in the woody part of the transgenic plant to be. In addition, the vector for introducing a target gene includes a cassette for introduction, and is a vector that allows a recombinant expression vector to be produced by facilitating introduction of a desired gene through site-specific recombination.

The expression vector or vector for introduction may be any vector which is available to the average person skilled in the art for expression in a plant by introducing a target gene, but may be used as a backbone. In particular, pK2GW7,0, pB2GW7,0, pKGWFS7,0, or pBGWFS7,0 may be used as the basic framework.

In a specific embodiment of the present invention, recombinant expression vectors PtrMX3-pKGWFS7,0 and PtrMX3-pBGWFS7,0 were prepared by introducing the PtrMX3 promoter using the SacI and SpeI restriction enzymes into the pKGWFS7,0 or pBGWFS7,0 vector 6). Also, PtrMX3-pK2GW7,0 and PtrMX3-pB2GW7,0, vectors for introducing the desired gene were prepared using the SacI and SpeI restriction enzymes in the pK2GW7,0 or pB2GW7,0 vector (Fig. 12).

In yet another embodiment, the present invention provides a bacterium comprising the recombinant expression vector.

The recombinant expression vector is as described above.

The bacterium may be any gene capable of producing a transgenic plant by introducing a recombinant vector into a plant, preferably Agrobacterium, and more preferably Agrobacterium tumefaciens (Agrobacterium tumefaciens) But is not limited thereto.

The term "Agrobacterium" in the present invention refers to a gram-positive bacterium that infects plant cells through the roots or stem wounds of the plant and inserts the gene of interest into the genome of the plant cell to transform the plant cell. It is a kind of carcinoma bacteria causing.

In yet another embodiment, the present invention provides a plant transformed with said recombinant expression vector.

The recombinant expression vector is as described above.

The term "transformation" in the present invention means that the DNA is introduced into the host and the DNA becomes replicable as a chromosome factor or by chromosome integration completion. Specifically, the present invention may mean introducing the recombinant expression vector into a host.

In the present invention, the term "plant" is selected to express a desired gene specifically in a woody part, and is not particularly limited to a species of plant if it has a cell wall and chlorophyll and has photosynthesized by independent nutrition. In addition, "transgenic plant" means a plant transformed with the above plant as a host.

In the present invention, transformation into a host cell includes any method of introducing the nucleic acid into an organism, cell, tissue or organ, and can be carried out by selecting a standard technique suitable for the plant, as is known in the art. Such methods include electroporation, electroporation, protoplast fusion, calcium phosphate precipitation, calcium chloride precipitation, agitation with silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate lipomethamine and drying / Mediated transformation methods, and the like, but the transformation method mediated by Agrobacterium is preferred.

In particular, the transgenic plants according to the present invention may be any plants, including herbaceous plants and woody plants, which are capable of expressing the PtrMX3 promoter and the target gene.

In particular, the herbaceous plant may be Oryza Sativa (rice), Zea mays (maize), Miscanthus species or Pennisetum purpureum, Eucalyptus species such as E. alba, E. albens, E. amygdalina, E. aromaphloa, E. baileyana, E. balladoniensis, E. bicostata, E. botryoides, E. brachyandra, E. brassiana, E E. breckistii, E. brockwayi, E. camaldulensis, E. ceracea, E. cloeziana, E. coccifera, E. cordata, E. cornuta, E. corticosa, E. crebra, E. croajingoleisis, E. curtisii, E. dalrympleana , E. deglupta, E. delegatensis, E. delicata, E. diversicolor, E. diversifolia, E. dives, E. dolichocarpa, E. dundasii, E. dunnii, E. elata, E. erythrocoiys, E. erythrophloia, E E. grandis, E. grandis, E. globulus subsp. bicostata, E. globulus subsp. globulus, E. gongylocarpa, E. grandis, E. grandis xurophylla, E. guilfoylei , E. gunnii, E. hallii, E. houseana, E. jacksonii, E. lansdowneana, E. latisinensis, E. leucophloia, E. leucoxylon, E. lockyeri, E. lucasii, E. maidenii, E. marginata, E. megacarpa, E. E. obtusiflora, E. occidentalis, E. optima, E. ovata, E. pachyphylla, E. coli, E. coli, E. coli, E. pellucida, E. pellitiana, E. pellitiana, E. petiolaris, E. pilularis, E. piperita, E. platyphylla, E. polyanthemos, E. populnea, E. preissiana, E. pseudoglobulus, E. pulchella, E. radiata, E. radiata subsp. E. spodulata, E. staphylococcus, E. spodulata, E. staphylococcus, E. coli, E. coli, E. coli, E. stoatei, E. tenuipes, E. tenuiramis, E. tereticornis, E. tetragona, E. tetrodonta, E. tindaliae, E. torquata, E. umbra, E. urophylla, E. vernicosa, E. viminalis, E. coli. wandoo, E. wetarensis, E. willisii, E. willisii subsp. falciformis, E. willisii subsp. willisii, E. woodwardii), populus species (for example, P. alba, P. alba P. grandidentata, P. alba P. P. tremula, P. alba P. P. tremula var. glandulosa, P. P. alba x P. tremuloides, P. balsamifera, P. balsamifera subsp. trichocarpa, P. balsamifera subsp. trichocarpa x P. deltoides, P. ciliata, P. deltoides, P. euphratica, P. euramericana, P. kitakamiensis, P P. grandifolia, P. maximowiczii, P. maximowiczii P. P. balsafera subsp. trichocarpa, P. nigra, P. sieboldii P. P. grandideiztata, P. suaveolens, P. szechuanica, P. tomentosa, P. tremula, P. tremuloides, P. tremuloides, P. wilsonii, P. canadensis, P. yunnanensis), conifers (for example, loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Picea glauca), redwood (Sequoia sempervirens), silver fir (Abies amabilis), pseudomonas spp., Pseudomonas spp., Pseudomonas spp. ; balsam oven (Abies balsamea) and cedar (e.g., Western red cedar (Thuja plicata), Alaska yellow-cedar (Chamecyparis nootkatensis)), and the like.

More specifically, it may be a herbaceous plant in an herbaceous plant, and may be, but is not limited to, poplar in a woody plant.

The term "Arabidopsis thaliana " in the present invention refers to a plant which belongs to the family Bressicaceae as a flowering plant and does not have agricultural importance but is very important for genetics and molecular biology research widely used as a model plant in modern botany. The Arabidopsis thaliana according to the present invention can be used as a representative plant of herbaceous plants, and specifically, it can be, but is not limited to, Arabidopsis thaliana, ecotype Columbia (Col-0).

In the present invention, the term "poplar" is a generic term for plants belonging to the broad-leaved willow tree willow tree and the squash tree. Specifically, in the present invention, poplar can be used as a representative plant of woody plants, and Populus alba x P. There is tremula. glandulosa. < / RTI >

In one embodiment of the present invention, in order to design a vector capable of inducing the expression of a target gene specifically in a woody part, a 35S promoter in a pKGWFS7,0 vector or a pBGWFS7,0 vector is designated SacI and SpeI The PtrMX3 :: GUS structure was constructed by replacing the promoter with the PtrMX3 promoter (SEQ ID NO: 3) using restriction enzymes. Then, the vector was introduced into Agrobacterium tumefaciens strain C58. Using the strain, the floral-dip method (Clough and Bent, 1998) and the leaf disk transformation-regeneration method (Horsch et al., 1985 ) Were transformed with Arabidopsis thaliana (ecotype Columbia (Col-0)) and poplar (Populus alba P, tremula var. Glandulosa)

Specifically, the transgenic plant according to the present invention may be introduced with the PtrMX3 promoter and a gene encoding a target protein operably linked to the PtrMX3 promoter so as to produce a desired protein in plants by overexpressing the target gene.

In one embodiment of the present invention, the organs of the transformed Arabidopsis thaliana transformed with PtrMX3 :: GUS of the present invention and the transgenic poplar of the present invention were analyzed in order to confirm whether the introduced gene was specifically expressed in the woody part of the plant (FIGS. 9). In another embodiment of the present invention, the degree of expression according to each stress was confirmed (FIG. 10 and FIG. 11) in order to confirm whether the gene expression varies depending on the internal and external stresses of the plant. As a result, in the transgenic Arabidopsis thaliana and transgenic poplar according to the present invention, the GUS protein was expressed specifically in woody tissues in all organs, and it was confirmed that GUS was expressed irrespective of the development stage of the plant, the internal and external stresses of the plant. Therefore, it was found that the PtrMX3 promoter of the present invention can be used to express the target gene specifically in the woody part of the plant, and thus it can be widely used in agriculture fields for various purposes.

In another embodiment, the present invention provides a method for producing a gene cassette comprising a step of introducing a target gene into a cassette of a target vector by site-specific recombination, And a manufacturing method thereof.

The above-mentioned position-specific recombination means that the entry vector and the target vector cause the LR reaction by the recombination enzyme, so that attR1 and attR2 of the target vector are replaced with attL1 and attL2 of the entry vector. By the LR reaction, the target gene of the entry vector may be introduced into the introduction vector, and the gateway cassette of the target vector may be deleted to produce an expression vector containing the desired gene.

The recombinant enzyme is a recombinant enzyme that replaces small double-stranded DNA fragments in DNA double helix, and is generally referred to as Recombinase (also referred to as Excisionase, Integrase), and may be, for example, LR clonase from Invitrogen, But is not limited thereto.

By the above method, an expression vector that specifically expresses a desired gene in a woody part can be easily prepared by introducing the target gene into the gateway cassette of the introduction vector.

In another aspect, the present invention provides a method for producing a transgenic plant expressing a target gene specifically in a woody part, which comprises transforming a plant with the recombinant expression vector.

In another embodiment, the present invention provides a method for producing a recombinant vector comprising: 1) transforming a plant with the recombinant expression vector; And 2) expressing a target gene in the transformed plant xylem tissue to produce a target protein.

The PtrMX3 promoter, the target protein, and the transgenic plant are as described above, and the transgenic plants of the present invention can be produced by the above method, and thereby the desired protein can be produced in the transgenic plant.

Specifically, the method may further comprise growing the transgenic plant in a soil or a medium. Cultivation of the transgenic plants in the present invention can be carried out according to well-known methods, and conditions such as cultivation temperature, cultivation time and pH of the medium can be appropriately adjusted.

The medium used should suitably meet the requirements of a particular transgenic plant. The medium may be a solid medium, a liquid medium or a double layer medium. Examples of the medium include water, colloid materials, agar, agarose, gelite, etc. may be used.

As inorganic nutrients, 15 elements, C, H, O, N, P, K, S, Ca, Mg, Fe, Mn, Cu, Zn, B and Mo, Carbohydrates, plant growth regulators, vitamins and the like may be added, and amino acids such as myo-inositol, glycine, L-glutamine and the like may be added.

Examples of the carbon source include glucose, fructose, mannose, ribose, xylose, sucrose, melibiose, cellobiose, lactose, amylose, carbohydrate, raffinose, sorbitol, mannitol, glycerol 1 and the like.

In a specific embodiment of the present invention, Arabidopsis thaliana (ecotype Columbia (Col-0)) and hybrid poplar clone BH1 (Populus alba P. tremula var. Glandulosa BH1) / 8 hours dark) or grown in half-strength MS-agar medium (Murashige and skoog, Sigma) containing the appropriate antibiotic and 2% sucrose for screening.

The transgenic plants produced by the above method can specifically express the target gene in the woody part to produce the target protein.

The plant-specific promoter of the present invention can induce superior wood-tissue-specificity and strong expression, independent of the developmental stage of the plant and external and internal environmental stresses.

FIG. 1 is a schematic diagram of sampling for gene expression analysis by tissue in poplar. FIG. A. Poplar stem grown for one year was used to separate and use each tissue. BP bark; DP is stem tissue; Ca is a cambium layer; DX is the conduit forming tissue; MX was a woody tissue and WS was used as a control throughout the stem. B. SL is apical mitotic tissue. C. ML is a mature leaf with vein removed. B and C were used as a control group without neck formation.
FIG. 2 shows a comparison of expression of transcripts of poplar tissues around the woody part MX. A total of 17,179 genes whose expression changes significantly were identified by comparing 10 kinds of poplar tissue transcripts, and they were plotted on the basis of the woody part (MX). That is, the gene whose expression is increased in the woody part is red, while the gene which is decreasing is expressed in blue. SM means summer stem and WN means winter stem.
Fig. 3 shows the expression of a gene specifically expressed in the woody part MX. Of the total 17,179 genes whose expression was significantly changed by comparing 10 kinds of poplar tissue transcripts, 116 genes specifically expressed in the woody part (MX) were identified using bioinformatics method . The graph on the bottom shows the comparison of the expression levels of the genes in each tissue, and the graph on the top shows logarithmic values of standardized gene expression levels.
Fig. 4 shows the expression pattern of Potri.013G007900.1 gene specific to woody part (MX) in tissue. The expression of Potri.013G007900.1 gene was visualized from the results of total transcript analysis by poplar organization. 6L, 6L and 6D represent Poplar oil plants grown 6 days at different light conditions, SL is shoot apical meristem & young leaf, YL is young leaf (young), Ca is cambium tissue, (b), (b) and (c), respectively. In the case of mature leaf, ML is mature leaf, BP is bark & phloem, DP is developing phloem, DX is developing xylem, MX is mature xylem, , And WN is the winter tissue of winter.
FIG. 5 is genetic information of PtrMX3 (Potri.013G007900.1) gene specific to woody part (MX), and PtrMX (Potri.013G007900.1) gene information obtained from Phytozome (www.phytozome.net). It is known that the gene is a gene whose function is unknown and that there is no intron.
6 shows a map of a recombinant gene vector used in the present invention. The PtrMX3 promoter was designed to regulate GUS gene expression by being located above the GUS gene. PKGWFS7,0 and pBGWFS7,0 were used as vector backbone. At this time, A was used for the development of Poplar transformants for the selection of kanamycin, and B was used for the development of Arabidopsis transformants for selection of basta.
FIG. 7 shows the activity of the PtrMX3 promoter specific to the woody tissue in the transgenic Arabidopsis thaliana (PtrMX3 :: GUS) plant. A. PtrMX3 :: GUS is a stem and root cross section of a transgenic Arabidopsis plant. Plants grown in soil for 50 days were used. Picture 1 is the cross section of the lower stem, Picture 2 is the cross section of the middle stem, and Picture 3 is the cross section of the root. Ep is an epidermis; Co is a cortex; IF is interfascicular fiber between vascular bundles; Xy is duct cell (xylem); Pi refers to pith cells. B. Pictures of other parts of the plant: leaves (1), flowers (2), fruits (3).
FIG. 8 shows the activity of the PtrMX3 promoter specific to the woody tissue in the transgenic Poplar (PtrMX3 :: GUS) plant. A. PtrMX3 :: GUS is a stem section of a transgenic poplar. Picture 1 is the cross section of the lower stem and Picture 2 is the cross section of the upper stem. Ep is an epidermis; Co is a cortex; IF is interfascicular fiber between vascular bundles; Pi refers to pith cells. B. It is a photograph of a leaf of a plant. It exhibits strong GUS activity only in the veins, and is not active in other tissues.
FIG. 9 shows the conduit-specific activity of the PtrMX3 promoter according to developmental stages of transformed Arabidopsis thaliana (PtrMX3 :: GUS) seedlings. A. 3 day old oil plants. The first photo shows the whole plant shape, the second is the cotyledon, the third is the hypocotyl enlarged photo, and the fourth is the root. B. Five day old oil plant. The first photo shows the whole plant shape, the second is the squamous tissue and the cotyledon, the third is the hypocotyl, and the fourth is the root. C. 7 day old oil plant. The first photo shows the whole plant pattern, the second is the hypocotyl and the cotyledon, the third is the hypocotyl, and the fourth is the root. D. 10 day old oil plant. The number 1 is the leaf shape, the number 3 is the enlarged hypocotyl, and the number 4 is the root.
FIG. 10 shows the change in the conduit-specific activity of the PtrMX3 promoter according to various environmental stresses in the transgenic Arabidopsis thaliana (PtrMX3 :: GUS). After 10 days of low temperature, dry, saline, and osmotic stress were treated with the transformed Arabidopsis thaliana (PtrMX3 :: GUS), the changes of the PtrMX3 promoter were examined. Mock is a control group that does not handle any stress.
Fig. 11 shows the change in the conduit-specific activity of the PtrMX3 promoter according to various plant hormone treatments in the transgenic Arabidopsis thaliana (PtrMX3 :: GUS). (PtrMX3 :: GUS) treated with various plant hormones (IAA, Kinetin or ABA) for 10 days were treated for 12 hours, and then the changes of the PtrMX3 promoter were examined. Mock is a control group with no hormones.
12 is a map of a recombinant gene vector using the PtrMX3 promoter. The PtrMX3-pK2GW7,0 and PtrMX3-pB2GW7,0 vectors were developed by replacing the 35S promoter of the existing pK2GW7,0 (for selection of kanamycin) and pB2GW7,0 (for selection of Basta) vector with the PtrMX3 promoter.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified in various forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  1: Screening of woody part-specific expression promoter and production of transgenic plants

1.1 Identification of wood-specific promoters

The present inventors isolated several tissues of Poplar tree (apical meristem, leaf, bark, stem tissue, cambium, ductal tissue, stem whole tissue) in order to search for a gene specifically expressed in the woody part of the plant And then mRNA was extracted from each tissue (Fig. 1).

The gene expression of each tissue was analyzed using Affymetrix GeneChip Poplar Genome Array. Poplar genome arrays have a total of 61,251 probe sets, which cover a total of 57,423 genes. Except for the repetitive genes, Poplar genome arrays can be used to study a total of 41,558 Poplar gene expression changes. A total of 10 tissues including poplar plant tissue, leaf, bark, stem tissue, cambium tissue, ductal tissue and stem whole tissue) and summer and winter stem tissues Transcript analysis was performed, and a total of 17,179 genes whose expression was significantly changed by each tissue were identified (Fig. 2). Of these 17,179 expression-altering genes, 116 genes having specific expression in the woody part were identified (FIG. 3), and the five genes having the strongest expression were selected to clone the respective promoters.

The promoter used was a sequence of about 1.5 kb in front of the ATG sequence, which is the translation initiation codon of each gene. In order to investigate the substantial wood-specific activity of each promoter in plants, a promoter :: GUS recombinant expression vector was prepared using the GUS gene as a reporter and introduced into Arabidopsis and Poplar trees to obtain transgenic plants Respectively.

Among the above five genes, the promoter of the Poplar Potri.013G007900.1 gene was particularly specific to the woody tissue and showed a very strong gene expression (Fig. 4). The expression of the gene in each tissue of poplar can be seen in a specific and strong expression peak in the wood cells, high expression in the summer period and low expression in the winter, so that the expression of the gene is strong in the active growth period Show. This Potri.013G007900.1 gene was named PtrMX3.

The PtrMX3 gene is a gene encoding a protein whose function is not known to date (FIG. 5, SEQ ID NO: 1 and SEQ ID NO: 2). In the present invention, the activity of the promoter was intensively examined.

To this end, in the present invention, a vector prepared by fusion of a GUS reporter gene to a PtrMX3 promoter is introduced to produce a transgenic poplar and a transgenic Arabidopsis thaliana, and the expression of the GUS gene in the transgenic plant is confirmed, whereby the tissue of the PtrMX3 promoter . The plant specimens used in the present invention and specific methods for producing PtrMX3 :: GUS transgenic plants are as follows.

1.2 Plant Specimens

Arabidopsis thaliana (ecotype Columbia (Col-0)) and hybrid poplar clone BH1 (Populus alba x P. tremula var. Glandulosa BH1) were used for the transformation experiments. The plants were cultured in MS-agar medium or soil at 23 占 폚, 16 hours / 8 hours dark condition.

1.3 PtrMX3  Promoter :: GUS  Production of Transgenic Plants

The genomic fragment (1524 bp) of the 5 'terminal end of the gene encoding Potri.013G007900.1 was isolated by PCR, and pBGWFS7,0 (for Arabidopsis transformation) or pKGWFS7,0 (poplar The genomic fragment was inserted in such a manner that the 35S promoter in front of the GUS reporter gene of the transformant was replaced with the PtrMX3 promoter. The nucleotide sequence of the replaced PtrMX3 promoter is shown in SEQ ID NO: 3.

Each of the vector constructs was first introduced into Agrobacterium tumefaciens strain C58 for plant transformation, thereby successfully producing a PtrMX3 promoter :: GUS transgenic plant.

Example  2 : PtrMX3  Identification of specific expression of woody part of promoter

2.1 GUS  Activity analysis

Histochemical staining was performed to confirm the activity of GUS (beta-glucuronidase) according to the tissues of the transgenic plants. 1 mM 5-bromo-4-chloro-3-indolylbeta-D-glucuronide, 100 mM phosphate buffer, 5 mM K ₃ [Fe (CN) ₆ ], 5 mM K ₄ [Fe (CN) ₆ ] and 10 mM EDTA at pH 7.0 for 18-24 hours at 37 [deg.] C. To better see the stained tissue, rinse the tissue specimen with ethanol for at least one hour at room temperature to remove chlorophyll. Then, expression of GUS protein was observed in each tissue of the transgenic plant.

2.2 Identification of woody part specific activity

GUS protein expression was observed in each tissue of transgenic plants prepared using the PtrMX3 :: GUS recombinant expression vector (FIG. 6). The GUS reporter protein degrades a substrate called X-Gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide) to form a blue precipitate, thus indicating the active position of the promoter in blue. As shown in the transgenic plants, the GUS activity (blue) was observed to be specific and very strong in the water tube cells expressed as Xy (xylem).

Specifically, in the case of Arabidopsis transgenic plants grown for 50 days in the soil, mature wood-specific and very strong GUS activity was observed in stem and root sections, and no activity was observed in other tissues (Fig. 7A). In addition, other organs of the same plant, such as leaves, flowers, fruits, and the like, did not show any activity except for a part of the vascular tissue in which water pipes exist (Fig. Similarly, in the case of the Poplar transformant in which the woody part was distinctly developed in the stem, unlike the Arabidopsis herbaceous plant, the GUS activity was strongly observed in the woody part (Fig. 8A). In leaf cells without woody part, there was no activity except vein (Fig. 8B).

As a result, the PtrMX3 promoter of the present invention was found to be specifically active in the woody part, and thus the target gene can be specifically expressed in the woody part using the gene expression vector containing the promoter.

Example  3: Depending on the stage of development of the plant PtrMX3  Identification of promoter activity

Next, in order to confirm whether the tissue-specific expression pattern by the PtrMX3 promoter changes depending on the stage of development of the plant, the activity of the PtrMX3 promoter according to the stage of development of the plant was examined.

As a result, GUS activity was specifically observed in young plants after germination, that is, from the third day of germination to tubercle tissue, and activity was observed at the same site on the 5th, 7th, and 10th day unchanged (FIG. 9). This suggests that the wood-specific activity of the PtrMX3 promoter appears from very young plants and does not change with developmental stages.

As a result, when the expression vector containing the PtrMX3 promoter of the present invention is used to specifically express the target gene in the woody part, the developmental stage of the target plant is not affected, and regardless of the development stage of the plant, And thus can be expressed as an antigen.

Example  4: Identification of promoter activity change by stress

On the other hand, the plants inevitably undergo various internal and external stresses during their growth. In order to determine whether the activity of PtrMX3 promoter changes depending on the stress, changes in the activity of PtrMX3 promoter were detected by treating with abiotic stress or plant hormone .

Specifically, seedlings of transgenic Arabidopsis thaliana (PtrMX3 :: GUS) grown in MS-agar medium were used, and the seedlings were pre-cultured in MS liquid medium for 12 hours, and plant hormones (1 μM IAA, kinetin or ABA) , MS medium containing 150 mM NaCl (salt stress) or 30 mM mannitol (osmotic stress), or at 4 ° C (cold stress). In addition, for the drought stress, the seedlings were dried in a laminar flow for 15 minutes, transferred to a standard MS liquid medium, and cultured for 12 hours. GUS activity analysis was performed according to the respective stresses.

First, in order to investigate the changes in PtrMX3 promoter activity when exposed to external environmental stress, PtrMX3 promoter activities observed in the woody tissues of the vaginal mucosa were examined by treating low temperature, high salt, high salinity and high osmotic stress on the dairy plants on the 10th day (Fig. 10).

On the other hand, there are five major hormones in plants (auxin, cytokinin, gibberellin, abscisic acid, ethylene) and these plant hormones play important roles in almost all parts of plant physiology. In order to simulate the internal stress of the plant, physiological changes, various plant hormones (IAA, kinetin, ABA) were treated on the 10th day old plants and the activity of the PtrMX3 promoter was examined. As a result, the PtrMX3 promoter activity, which is observed only in the mural tissues of the vagina, as in the external environmental stress, was not found to be significant despite each plant hormone treatment (FIG. 11).

Taken together, these results indicate that the activity of the PtrMX3 promoter does not change with the development stage of the plant or with various internal and external stresses, and is consistently specific to the woody tissue and has strong activity. Therefore, the expression vector containing the PtrMX3 promoter of the present invention has an excellent effect of strongly expressing the gene of interest in the plant-specific transgenic plant regardless of the development stage of the plant or various internal and external stresses.

Example  5: Utility vector manufacturing

In order to specifically express the exogenous useful gene using the PtrMX3 promoter developed in the present invention, it is necessary to prepare a recombinant gene vector (for example, PtrMX3 :: GENE). In order to facilitate the production of the recombinant gene vector, a utility vector containing the PtrMX3 promoter was prepared.

The cloning method using a general restriction enzyme and a ligase is time consuming and may often have various problems. However, the gateway cloning method uses a nucleotide sequence-specific recombination method, and the nucleotide sequence of the gene to be cloned Regardless of how it is known in a very efficient way. The existing vector for the gateway is connected to the 35S promoter and the gateway cassette, and the target gene is cloned into the gateway cassette.

In the present invention, PtrMX3-pK2GW7,0 (for kanamycin screening) and PtrMX3-pB2GW7,0 (for basta screening) vectors in which the 35S promoter was replaced with the PtrMX3 promoter were prepared (Fig. 12). A transformant expressing a target gene specifically in the woody part of a plant can be easily prepared using the vectors.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the spirit and scope of the present invention as defined by the appended claims.

<110> University-Industry Cooperation Group of Kyung Hee University <120> A XYLEM-SPECIFIC PROMOTER AND USES THEREOF <130> KPA141323-KR <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 417 <212> DNA <213> Populus trichocarpa <400> 1 atgatgaaga cgaccgtcgc agtagtattt gtgctagggc tggcattctt ggatcttcaa 60 gttgatgcta aaaggcttct tttgaaggaa atcaaggcag aaaaaactga tgataagcct 120 ttgtctaatg tgcagcagga tggtaaacta gatgctgtta acaatgctgg aactgatgtt 180 aagccgaata atcaaccagg aaacgttggc acctatggta atccagtgac aggctcagtg 240 cccgcagttg ataccaagaa tgataatgca actagcagcc cgtctactag tgacaacaag 300 ggcagcactg acgatgaggc caacagcagt tatgggaact atggcaatcc ttctggatca 360 tctactgata ctcaccattc ctttaccaat gattgccagc ctaagaaagg ttgttga 417 <210> 2 <211> 138 <212> PRT <213> Populus trichocarpa <400> 2 Met Met Lys Thr Thr Val Ala Val Val Phe Val Leu Gly Leu Ala Phe   1 5 10 15 Leu Asp Leu Gln Val Asp Ala Lys Arg Leu Leu Leu Lys Glu Ile Lys              20 25 30 Ala Glu Lys Thr Asp Asp Lys Pro Leu Ser Asn Val Gln Gln Asp Gly          35 40 45 Lys Leu Asp Ala Val Asn Asn Ala Gly Thr Asp Val Lys Pro Asn Asn      50 55 60 Gln Pro Gly Asn Val Gly Thr Tyr Gly Asn Pro Val Thr Gly Ser Val  65 70 75 80 Pro Ala Val Asp Thr Lys Asn Asp Asn Ala Thr Ser Ser Ser Thr                  85 90 95 Ser Asp Lys Gly Ser Thr Asp Asp Glu Ala Asn Ser Ser Tyr Gly             100 105 110 Asn Tyr Gly Asn Pro Ser Gly Ser Ser Thr Asp Thr His Ser Phe         115 120 125 Thr Asn Cys Gln Pro Lys Lys Gly Cys     130 135 <210> 3 <211> 1524 <212> DNA <213> Artificial Sequence <220> <223> PtrMX3 promoter <400> 3 acgcatgcct tcacttccat ttttttatta aagactaaat tttttgacct ccgttggaaa 60 agttggacta aacttgagat gggttttgac ccttttggat tggaagtaaa aaagaaaaga 120 gatttagcaa atctggggct tgatagagaa gatggagggg atagtagttt catggccatg 180 gcttggcctt tcattttttt cccttttttg gtggttaaac tgattctgtt ttgagtggcg 240 ggtactcggt ggcctgtttt agttctattg gattttgtta gaacaaaatt aatccttaat 300 gttttatatt atgaaaaact ggtcccttct ggagtttttt ttttttttaa gggaaattgg 360 tagaagtgag tgagtttggt ttctctatct ttttagtggt ggagaaatgg ttttgatttt 420 gctaagttta tttgtggggc ttcgaataat ttagtgcttt atccatttgg tgaccactct 480 tcgtgaccca tcaaagggta tgctttttga aatttcattc tgattttttt tgtttaaatt 540 taatttttta tatattatttt tagattattt ttaaaaagta aaaaatatca ttttaaaata 600 tttttaaaca aaaaatattt taaaaataac taaaatattc tcaaataccc tataattaac 660 ctattgggtg ctttatcacc actctttttg atgggtgaaa gggtatggac ctggctggtt 720 aagtgtatga gctcatgcta gggttagccc catacttata tggtaacttg ggttgtaatg 780 agcttagata gcccaaacag gtccaattca gcttgagagc ctgattttct caacattttt 840 cagaagcgaa gaacagtttt tagctccatg tttacccctt agattactta tcttagcaat 900 cagatcaact ttatgatccc ataattacaa tttacctaga gcatatgcag gatgcatgct 960 tgtctgtatt caaattatta atgatcttat tgttatgtca tcaaccaatt aacatgccat 1020 tgctagctgg tagattcata gtgcataaac tgtatatttt ggtgctgcac ttgctgagcc 1080 ctacaacgca aacatcatta agcctcaata taaacaaaac caagtaaaaa tctcaaccct 1140 ttattcagat tatctgatct gtttttcacc cttgttcata attactactg tatctggtgg 1200 cccctttcaa gcacacctga gtgtggcctc accaataagg ccagaagatt gtgagtttgt 1260 aatcactcca ctacttcatt gtgctcacat tttaatctta gtttctcact aatcatgatt 1320 attataaaca tgtttcttct ataaatttgc ccccttgatg caatgttaat caaccaatga 1380 tcgcactaga gaagggatat aaggtacgta agagtgggtg gaaataccat ttttagttaa 1440 actttcagtt tagaagtaaa ttacttattt aatcttaata cctctgcata cttggataat 1500 ctgctgttga ttgaaaataa cagg 1524

Claims (15)

A wood-specific promoter of a plant, which is composed of the nucleotide sequence of SEQ ID NO: 3.
2. The promoter of claim 1, wherein the promoter specifically expresses a gene of interest operably linked thereto in a xylem.
The promoter according to claim 1, wherein the plant is poplar or Arabidopsis.
A plant cell specific expression cassette of a gene of interest, comprising the promoter of claim 1 and a target gene operably linked thereto.
A cassette for introducing a target gene for producing a plant-specific gene expression cassette of a gene of interest, comprising the promoter and the gateway cassette of claim 1.
A recombinant expression vector comprising the expression cassette of claim 4.
A vector for introducing a target gene comprising a cassette for introducing a gene.
7. The vector according to claim 6, wherein the vector is a backbone of pK2GW7,0, pB2GW7,0, pKGWFS7,0 or pBGWFS7,0.
A bacterium comprising the recombinant expression vector of claim 6.
10. The bacterium of claim 9, wherein the bacterium is Agrobacterium tumefaciens .
A plant transformed by the recombinant expression vector of claim 6.
12. The plant according to claim 11, wherein the plant is poplar or Arabidopsis.
[Claim 7] The method according to claim 7, which comprises introducing a target gene into a gateway cassette of a vector for gene introduction using a site specific recombination.
A method for producing a transgenic plant expressing a gene of interest specifically for a woody plant, comprising the step of transforming the plant with the recombinant expression vector of claim 6.
1) transforming a plant with the recombinant expression vector of claim 6; And
2) expressing the desired gene in the xylem tissue of the transformed plant to produce the desired protein.

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