KR101820925B1 - Root tissue-specific promoter - Google Patents

Abstract

The present invention relates to root-tissue specific promoters of plants. The root tissue specific promoters of the plants of the present invention have an advantage of inducing a foreign target gene to be specifically expressed only in the root tissue without expressing the foreign target gene in other tissues of the plant. Therefore, the root-tissue specific promoters of plants can be effectively used for the development of transgenic plants intended to produce useful proteins in root tissues, thereby being usefully used in a trait improvement industry related thereto.

Description

Root tissue-specific promoter < RTI ID = 0.0 >

The present invention relates to root-tissue specific promoters of plants.

Crop molecular breeding can use all kinds of genes as a material and it is possible to control the effect of breeding infinitely by dragging the breeding technique which was possible only by the existing genome unit as a gene unit, Technology. The cultivation area and the cultivation amount of genetically modified crops using this crop molecular breeding technique are continuously increasing all over the world, and the global market size of the commercialized genetically modified new person is from $ 4 billion in 2002 to $ 10 billion in 2010 And 15% of the global seed market, which is 15% of the US $ 30 billion market. Soybean, corn, cotton, and rapeseed are the main markets. In order to maximize the effect of genetically modified crops, the expression of exogenously inserted genes must be finely regulated by the developmental stage and tissue of the plant, and various promoters should be developed for this purpose. A promoter is a regulatory gene that regulates the expression of a gene. When a specific promoter is used, useful genes can be expressed at a suitable time or at a specific site of a plant, which is an important technique in the development of new varieties using biotechnology. In particular, the development of promoters that induce tissue-specific expression will be very useful and important. Therefore, competition for securing relevant intellectual property rights is being widely carried out not only in Korea but also around the world.

Since the early 1980s, research has been conducted on promoters that regulate the expression of plant genes. It is suggested that the promoter of cauliflower mosaic virus induces strong expression in whole plant tissues (Hohn et al., 1982, Curr. Topics Microbiol. Immunol. 96: 193-236) (Odell et al., 1985, Nature 313: 810-812) and demonstrated strong expression in plant bodies (Sanders et al., 1987, Nucleic Acids Res. 15: 1543-58). The CaMV 35S (patent number: JP1993192172-A1) promoter has since become the universal promoter most commonly used in plants.

On the other hand, studies on the expression of a tissue-specific promoter inducing specific tissue-specific expression of plants have been progressed steadily, and specifically, flower-tissue specific promoters (van der Meer et al., 1990, Plant Mol. Biol. 15: Plant Molecular Biology, 66: 379-388; Verdonk et al., 2008, Plant Biotechnology Journal, pp. 95-109; Ruiz-Rivero and Prat, 1998; Plant Mol. Biol. 36: 639-648; (Van Tunen et al., 1990, Plant Cell 2: 393-401; Kato et < RTI ID = 0.0 > al., & , 2010, Plant Molecular Biology Reports 28: 381-387), seed specific promoters (Bustos et al., 1989, Plant Cell 1: 839-853; Kridl et al., 1991, Seed Sci Res, 1: 209-219; Plant Biotechnology Journal 6: 679-693; Chung et al., 1999; Welham and Domoney, 2000; Plant Sci. 159: 289-299; Plant Mol. Biol. 40: 1-12; Furtado et al. et al., 2008, Plant Cell Reports, 27: 29-37).

In addition to the stem supporting the rooted plant, there is a supporting action to support the plant, an absorption action to absorb the water and inorganic nutrients in the soil, and a storage action to store the nutrients resulting from the photosynthesis of the plant in the roots. Importance of these roots It is important to keep the roots of the crops of good traits healthy during the growing process, especially the root crops are closely related to yield and yield. Therefore, there is a continuing need for research that can induce the expression of a target gene using a promoter specifically expressed in root tissues and the promoter.

The present inventors have found that results were transformed to after making a vector comprising while trying to identify a promoter that is expressed specifically only in root tissue of the plant, determine the Arabidopsis promoter DCD1 the pole At2g32910 gene, and this end, the plant the vector , But not in other tissues such as flowers, stems and leaves, and the DCD1 promoter is specifically expressed only in the root, and the DCD1 promoter of the present invention induces the expression of the target gene to be expressed only in the root, Thereby completing the invention.

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

It is also an object of the present invention to provide a transforming expression vector comprising the promoter.

It is also an object of the present invention to provide a root-tissue-specific transformed cell of a plant transformed with said expression vector.

It is also an object of the present invention to provide a method for producing root tissue-specific transgenic plants.

It is also an object of the present invention to provide a method for specifically expressing a foreign gene in plant root tissue.

In order to solve the above problems, the present invention provides a root tissue-specific promoter of a plant comprising the nucleotide sequence shown in SEQ ID NO: 1.

The present invention also provides a transforming expression vector comprising the promoter.

The present invention also provides a root-tissue-specific transformed cell of a plant transformed with said expression vector.

(1) preparing a vector comprising a promoter comprising the nucleotide sequence shown in SEQ ID NO: 1; And (2) introducing the vector into a plant or plant cell and culturing the plant.

(1) preparing a vector comprising a promoter and a foreign gene comprising the nucleotide sequence shown in SEQ ID NO: 1; And (2) introducing the vector into a plant. The present invention also provides a method for specifically expressing a foreign gene in plant root tissue.

The root tissue specific promoter of the plant of the present invention induces a foreign target gene to be specifically expressed only in the root tissue without being expressed in other tissues of the plant. Therefore, it can be effectively used in the development of transgenic plants for producing the desired useful protein in root tissues, and can be usefully used in the related-arts and the like.

1 is a schematic diagram showing an expression vector containing DCD1 which is a promoter of the Arabidopsis At2g32910 gene.
FIG. 2 shows the result of RT-PCR analysis of DCD1 gene expression in each tissue of Arabidopsis thaliana transformed with the vector of the present invention (FL is a flower, ST is a stem, CL is a stem leaf, RL is a rosette leaf, RO means roots and SE means pods).
FIG. 3 is a diagram showing the results of analysis of DCD1 gene expression by each tissue of Arabidopsis thaliana transformed with the vector of the present invention by histochemical staining (A is germination day 7 plant, B is germination day 20 plant, C FL is the stem, CL is the stem leaf, RL is the rosette leaf, RO is the root, B is the root extension, D is the cross-section of the root of C, E is the germination 4 weeks tea plant, Means roots).

The present invention provides a root tissue-specific promoter of a plant comprising the nucleotide sequence shown in SEQ ID NO: 1.

In the present invention, a "promoter" is a gene region located upstream of an encoding site, which generally includes a site at which transcription is initiated, and is usually a TATA box that regulates gene expression, a CAAT box site, And an enhancer that promotes the expression of almost all genes regardless of the site and direction of the gene that affects 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. For the purpose of the present invention, the promoter according to the present invention is a DCD1 promoter of the Arabidopsis thaliana At2g32910 gene that is expressed specifically in root tissue, and the promoter can induce the expression of the target gene in the root tissue. The DNA sequence (SEQ ID NO: 1) of the promoter is characterized in that it is derived from -1 to -482 bp from the translation initiation site of the coding sequence of the Arabidopsis At2g32910 gene.

The nucleotide sequence variants of SEQ ID NO: 1 are included within the scope of the present invention. Although the nucleotide sequence of SEQ ID NO: 1 of the present invention and the functional equivalents of the nucleic acid molecules constituting it, for example, some nucleotide sequences of SEQ ID NO: 1 have been modified by deletion, substitution or insertion , A nucleotide sequence of SEQ ID NO: 1, and variants capable of functionally equivalent to a nucleic acid molecule constituting the nucleotide sequence. Specifically, it may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 have. "% Of sequence homology" for a nucleotide sequence is determined by comparing two optimized sequences with comparison regions, and some of the nucleotide sequences in the comparison region are referred to as the reference sequence (addition or deletion) (I. E., Gap), as compared to < / RTI >

The plants include vegetable crops including Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onion, onion and carrot; Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Special crops including ginseng, tobacco, cotton, sesame seed, sugar cane, beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots, and bananas; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; And forage crops including raglass, red clover, orchardgrass, alfalfa, tall fescue, and penny al raglas, and the foreign gene of interest is introduced by using the DCD1 promoter of the present invention, But is not limited to, the roots of plants that can be derived.

The present invention also provides a transforming expression vector comprising the promoter.

In the present invention, the term "vector" means a gene product containing a base sequence of a gene operably linked to a suitable regulatory sequence so as to express the gene of interest in a suitable host. A promoter that can be used, any operator sequence to control such transcription, and sequences that control the termination of transcription and translation.

In the present invention, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. For example, a nucleic acid sequence encoding a promoter and a protein or RNA may be operably linked to affect the expression of the coding sequence. The operative linkage with the recombinant vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and linkage are made using enzymes generally known in the art.

In the present invention, the expression "expression of a desired gene" may mean expression of the desired gene to produce a protein encoded by the desired gene. In the present invention, a method of expressing a target gene may be a method of expressing a protein encoded by the target gene by culturing a transformant (host cell) transformed with a vector containing the target gene, The final product of the involved biosynthetic pathway can be produced.

The vector of the present invention may also include a selection marker, a foreign gene, a promoter, and a terminator.

Markers that can be used in the present invention are nucleic acid sequences having characteristics that can be generally selected by a chemical method, including all genes capable of distinguishing transformed cells from untransformed cells.

The vector of the present invention is not particularly limited as long as it is replicable in cells, and any vector known in the art can be used. Therefore, plasmids derived from Escherichia coli (pBR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5, etc.) and yeast-derived plasmids (YEp13 and YEp24 And YCp50), and Ti plasmids, and there are plant virus vectors, and binary vectors such as pCHF3, pPZP, pGA and pCAMBIA series can be used. However, those skilled in the art can use any vector as long as it can introduce the promoter of the present invention and the base sequence of the target gene to be expressed in the promoter into the host cell, but preferably the vector pBI101 is used. Wherein the vector is a vector containing the GUS reporter gene, GUS (beta-glucuronidase) reporter gene is an example of a foreign gene, so as to position the DCD1 promoter of the present invention in front of the foreign gene, DCD1 promoter :: GUS (hereinafter referred to as Pro DCD1 ). The GUS reporter gene may be substituted with a useful foreign gene for another purpose.

The foreign gene may be any gene which desires to be expressed in the root tissue of the plant, and is located behind the promoter region in a plant-specific expression vector of the present invention, and may be expressed and fused with a reporter gene, if necessary.

In the present invention, the term "transformation" refers to the introduction of DNA into cells. The cells are termed "host cells ", which may be prokaryotic or eukaryotic cells. Representative prokaryotic host cells include various strains of E. coli. The introduced DNA is usually in the form of a vector containing a fragment into which the DNA is inserted. The introduced DNA sequence may be from the same species as the host cell, or from a different species than the host cell, or it may be a hybrid DNA sequence containing some DNA and some external DNA originating from the host species.

Such plants include whole plants, plant organs (e.g., leaves, stems, flowers, roots, etc.), seeds and plant cells (including tissue culture cells) and their progeny. The classes of plants that can be used in the method of the present invention are broad, such as the class of higher plants generally applicable to the transformation technology, and certain lower plants such as algae. This includes plants of varying levels of purity, including polyploids, diploids and haploids.

For example, the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. (Klein et al., 1987, Nature 327, 70), infiltration of plants or the transformation of Agrobacterium species by transformation of mature pollen or microparticles And infection by (non-integrative) viruses in Agrobacterium tumefaciens mediated gene transfer (EP 0 301 316).

The optimal procedure for transformation of the plant by the Agrobacterium vector varies depending on the kind of plant to be transformed. Representative methods for Agrobacterium-mediated transformation include transformation of excreted pieces of hypocotyl, shoot tip, stem, leaf or flower tissue derived from sterile seedlings and / or small plants. Such transformed plants can be propagated by ovarian reproduction or by cell or tissue culture. Agrobacterium-mediated transformation has previously been described for a number of different kinds of plants and methods for such transformation can be found in literature known in the art. As the Agrobacterium strain for plant transformation that can be used in the present invention, any strain commonly used can be used.

The present invention also provides a root-tissue-specific transformed cell of a plant transformed with said expression vector.

The expression vector may be introduced into host cells cultured using well known techniques such as infection, transfection, transfection, electroporation and transformation. Representative examples of the host include, but are not limited to, plant cells, bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells. Any plant cell can be used as "plant cell" used for transformation of a plant. The plant cell may be any type of cultured cell, cultured tissue, cultured organ or whole plant, preferably cultured cell, cultured tissue or culture organ, and more preferably cultured cell. "Plant tissue" refers to a tissue of a differentiated or undifferentiated plant, such as, but not limited to, stem cells, leaves, cancer tissues, and various types of cells used in culture, such as single cells, protoplasts, Callus tissue.

(1) preparing a vector comprising a promoter comprising the nucleotide sequence shown in SEQ ID NO: 1; And (2) introducing the vector into a plant or plant cell and culturing the plant.

In the present invention, "cultivation" means that the plant or plant cell is grown under moderately artificially controlled environmental conditions.

The plant cells can be grown in a conventional medium. The culture medium may contain nutrients required for culturing a target plant cell, that is, a plant cell to be cultured, and may be a mixture with a specific purpose added thereto. The medium is also referred to as an incubator or a culture medium, and is a concept including both natural medium, synthetic medium and selective medium.

(1) preparing a vector comprising a promoter and a foreign gene comprising the nucleotide sequence shown in SEQ ID NO: 1; And (2) introducing the vector into a plant. The present invention also provides a method for specifically expressing a foreign gene in plant root tissue.

Hereinafter, the present invention will be described in detail by way of examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention in any way to the scope of the invention as defined by the appended claims. It will be obvious.

Example 1 . Arabian At2g32910  A promoter of a gene DCD1 (Development and cell death domain containing protein 1) promoter cloning

Arabidopsis thaliana ecotype Columbia) S primer (5'-CATGTCGACGTAAGAGTTCTTCGAGCAGACTCTAGATTC-3 'containing the Sal (5' end) and Sma (3 'terminal) at both ends by a total of gDNA (genomic DNA) as the template of SEQ ID NO: 2) and PCR method was used for 482 bp of the At2g32910 gene promoter DCD1 using the AS primer (5'-CATCCCGGGTTTCAAGGAACAAGCAGATTTCAAAGC-3 ', SEQ ID NO: 3). Specifically, the amplification was performed at 95 ° C. for 2 minutes After that, PCR was performed 33 times at 95 ° C for 20 seconds, 57 ° C for 20 seconds, and 72 ° C for 50 seconds. The PCR products were digested with restriction enzymes and inserted into pBI101 vector, a plant-transforming binary vector containing GUS (beta-glucuronidase) between the left boarder and the right boarder 1, which is shown in Fig. The sequencing of the clones confirmed that the correct nucleotide sequence was cloned, and the entire nucleotide sequence of the DCD1 promoter was represented by SEQ ID NO: 1.

Example 2. DCD1  Promoter Arabidopsis transformation

The vector shown in Fig. 1 prepared in Example 1 was introduced into Agrobacterium GV3101 by a freeze-thaw method (An, G. 1987, Methods in Enzymology). The transformed Agrobacterium was transformed into Arabidopsis thaliana by the flower dipping method (Clough and Bent, 1998, The Plant Journal). The transformed Arabidopsis thaliana was screened in a plant MS (Murashige and Skoog) medium containing 50 mg / ml of kanamycin to obtain a transformed plant.

Example 3: Transgenic Arabidopsis thaliana < RTI ID = 0.0 > DCD1  Expression analysis of promoter

In the tissue of the Arabidopsis transformed with the vector prepared in Example 1, DCD1 Real-time PCR was performed to confirm gene expression, DCD1 Chemical staining was performed to confirm expression of the promoter.

3-1. Transgenic Arabidopsis of each tissue DCD1  Real-time PCR for gene expression analysis

Specifically, rosette leaves, cauline leaf, stem, flower and silique of the 4 week-old transformed Arabidopsis obtained in Example 2 were sampled by tissue To extract RNA. (5'-GACATACATGGTTTCTCCAGGTAGA-3 ', SEQ ID NO: 4) and AS primer (5'-CCATATCCAAGATACATTAAACATCAGATAAATTC-3', SEQ ID NO: 5) at 95 ° C. for 5 minutes . After the initial denaturation, real-time PCR was performed at 95 ° C for 10 seconds, 54 ° C for 10 seconds, and 72 ° C for 20 seconds to observe the expression of the DCD1 gene. The results are shown in Fig.

As shown in Figure 2, DCD1 The expression of the gene was confirmed to be high.

3-2. Transgenic Arabidopsis of each tissue DCD1  Histochemical staining for expression analysis of promoter

Specifically, each tissue of the germinated 7th day, 20th day and 4th week transformed Arabidopsis thaliana obtained in Example 2 was obtained. Then, each of the above tissues was treated with 1 mM of 5-bromo-4-chloro-3-indolyl-β-glucuronidase, 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, After immersing in a solution containing 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 at 37 ° C for 12 hours, the solution was removed, and 100% And the activity of the transformed Arabidopsis GUS (beta-glucuronidase) was analyzed. The results are shown in Fig.

As shown in FIGS. 3A and 3B, it was confirmed that GUS was specifically expressed in the root tissues of Arabidopsis thaliana transformed with germination day 7 (A) and germination day 20 (B). Further, as shown in C and D in Fig. 3, a photograph (C) showing an enlarged root structure of the Arabidopsis thaliana transformed with germination for 20 days and a cross-sectional view (D) . In addition, as shown in E and F of Fig. 3, roots (RO), flowers (FL), stems (ST), stem leaves (CL) and rosette leaves (RL) And GUS, the expression of GUS was specifically detected only in root tissues.

Thus, through the above-mentioned series of results, it was confirmed that the DCD1 promoter represented by SEQ ID NO: 1 is a root-specific promoter.

<110> Korea University Research and Business Foundation Root tissue-specific promoter <130> 1-76 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 482 <212> DNA <213> Unknown <220> <223> DCD1 promoter <400> 1 gtaagagttc ttcgagcaga ctctagattc gatttgtttt tttccttttc gggtttgttt 60 caaatcaatc tcaatttcag ggtaaccaaa gtttgagctt attcattttg attctgcccg 120 gatctctata catgtcaatt acagtctttt tacattttct tgatttggaa accttttcgg 180 gatttaattg attagcttaa gattacaagt gatgaaaaaa aagacagaga ctttagcaaa 240 tgttttctct catctcttaa ttgctgtctt aattacaatc ttcttgtttc agttactgtt 300 ttgtaagaca ttgacattat aatctacttg tttggagatt ggaattatga gaagagaatc 360 tttctgatgc caaatgtcat tgaataatca tttgctaatg tttcttttac catatgattc 420 atgatctctt tttcgtttta tgttgcagcg actaagcttt gaaatctgct tgttccttga 480 aa 482 <210> 2 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer 1 <400> 2 catgtcgacg taagagttct tcgagcagac tctagattc 39 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 <400> 3 catcccgggt ttcaaggaac aagcagattt caaagc 36 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> forward primer 2 <400> 4 gacatacatg gtttctccag gtaga 25 <210> 5 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 <400> 5 ccatatccaa gatacattaa acatcagata aattc 35

Claims (7)

A root tissue-specific promoter of a plant consisting of the nucleotide sequence shown in SEQ ID NO: 1. The method according to claim 1,
The plants include vegetable crops including Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onion, onion and carrot; Food crops including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; Special crops including ginseng, tobacco, cotton, sesame seed, sugar cane, beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots, and bananas; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; And feed crops including rice grains, red clover, orchardgrass, alfalfa, tall fescue, and penny al raglas; and the root tissue specific promoter of the plant. A transforming expression vector comprising the promoter of claim 1. A root-tissue-specific transformed cell of a plant transformed by the expression vector of claim 3. A transformed plant transformed with the vector according to claim 3 or the transformed cell according to claim 4. (1) preparing a vector comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1; And
(2) introducing the vector into a plant. (1) preparing a vector comprising a promoter and a foreign gene consisting of the nucleotide sequence shown in SEQ ID NO: 1; And
(2) introducing the vector into a plant; and (2) introducing the vector into a plant.

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