KR101211957B1 - The promoter from Brassica napus and method for using thereof - Google Patents

Abstract

The present invention relates to a rapeseed seed specific promoter, and to a promoter capable of inducing expression of a gene located downstream of a promoter only in a rapeseed plant seed and a method for accumulating a large amount of a target protein using the promoter in a plant seed. .
The promoter having the plant seed specificity of the present invention can be used for the purpose of specifically expressing the protein of interest in the plant seed, and can not only contain an excessive amount of the useful protein having a medicinal use or physiological activity in the plant seed, as well as fatty acids. Improved plant varieties can be used to industrially use seed oil and improve the productivity of seed plants, including rapeseed.

Description

Rape-derived seed specific expression promoter and its use {The promoter from Brassica napus and method for using pretty}

The present invention relates to a rapeseed-derived seed specific expression promoter and its use, and more particularly to a rapeseed-derived seed specific expression promoter, a recombinant plant expression vector comprising the promoter, and a method of producing a target protein using the recombinant plant expression vector. The present invention relates to a method for producing a transformed plant using the recombinant plant expression vector, a transformed plant prepared by the method, and a method for confirming whether the plant is transformed using the promoter.

Rapeseed (Brassica napus) is a cruciferous plant, grown mainly in the southern part of Korea, sowing in autumn and flowering and fruiting in the following spring, leaves harvested in late winter or early spring, and seeds harvested in early summer. This is done. Rapeseed oil (canola oil), which is extracted from the seeds of rapeseed, is widely used for food, and the leaves of rapeseed are used for cooking as herbs.

A promoter is a genome region that is linked to the upper side of a gene, and controls a structural gene linked thereto to be transcribed into mRNA. Promoter is activated by the combination of a number of general transcription factors (general transcription factors), it has a base sequence such as TATA box, CAAT box, which regulates gene expression. Proteins necessary for the basic metabolism of the living body must maintain a constant concentration in the cell, so the promoters linked to these genes are always activated by the action of the general transcription factor.

Promoters used in the transformation of plants are usually promoters that are mainly expressed in the entire tissues or cells of the plant. These promoters lack tissue-selectivity or organ-selectivity, resulting in delayed plant growth by the expression of transformed selection markers in whole plants. In addition, due to the nature of the promoter, the expression of the introduced gene is limited to the target organ, and can not be expressed sufficiently, resulting in an inferior economic efficiency of the transformant. However, currently, studies on promoters isolated from plants having organ-specific characteristics are insufficient.

Promoters reported to be involved in plant tissue specific or organ specific expression include the promoter of the ZmC5 gene, a corn pollen specific promoter of Korean Patent Publication No. 2001-41129, and the plant of Korean Patent Publication No. 2001-66718. There is a promoter of trehalase gene isolated from Arabidopsis having covariate specific gene expression, there is a promoter of cataraze A (CatA) gene having anther and pollen specificity of rice of Korea Patent Publication No. 2001-106119 , The promoter of the oleosin gene having flax seed specificity and the promoter of legumin-like seed storage protein of Korean Patent Publication No. 2002-57950, and the Cv20oxP promoter having the seed specificity of watermelon of Korean Patent Publication No. 2002-93028 Plant seed derived from sesame seeds of Korean Laid-open Patent Publication No. 2004-41483 There is a promoter of the microsome oleic acid unsaturated gene.

The present invention relates to a rapeseed seed specific promoter, and to a promoter capable of inducing expression of a gene located downstream of a promoter only in a rapeseed plant seed and a method for accumulating a large amount of a target protein using the promoter in a plant seed. .

The present invention provides a rapeseed seed specific promoter for plant gene transformation, and more specifically provides a rapeseed seed specific promoter having a nucleotide sequence of SEQ ID NO: 1.

The present invention relates to a specific promoter derived from rapeseed, specifically, the promoter is a promoter of the 12S globulin gene of SEQ ID NO: 1.

In addition, variants of such promoter sequences are included within the scope of the present invention. A variant is a nucleotide sequence that changes in nucleotide sequence but has similar functional properties to the nucleotide sequence of SEQ ID NO: 1. Specifically, the promoter may include at least 70%, at least 80% or at least 90%, or at least 95% sequence homology with the base sequence of SEQ ID NO: 1, respectively.

The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).

The present invention provides a recombinant plant expression vector comprising the promoter according to the present invention. As an example of the recombinant plant expression vector, pBGWSF7.0 vector used in the examples may be mentioned, but is not limited thereto.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and a suitable nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

Preferred examples of plant expression vectors are Ti-plasmid vectors which, when present in a suitable host such as Agrobacterium tumerfaciens, can transfer part of themselves, the so-called T-region, into plant cells. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as disclosed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector may comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotics such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol Resistance genes include, but are not limited to.

In the plant expression vector according to an embodiment of the present invention, the terminator may use a conventional terminator, and examples thereof include nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminators of the octopine gene of bacterium tumefaciens, but are not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells.

In a recombinant plant expression vector according to one embodiment of the present invention, the plant expression vector may be prepared by operably linking a target gene downstream of the promoter of the present invention. In the present invention, "operably linked" refers to a component of an expression cassette that functions as a unit for expressing a heterologous protein. For example, a promoter operably linked to heterologous DNA encoding a protein promotes the production of functional mRNA corresponding to the heterologous DNA.

The gene of interest may be any kind of gene, for example, encodes a protein of medical utility such as enzymes, hormones, antibodies, cytokines, or accumulates a large amount of nutrients that can enhance the health of animals including humans. A protein capable of encoding the gene may be, but is not limited thereto. In addition, the target gene may be a gene present in a host into which the recombinant plant expression vector is introduced or may be a foreign gene.

In one embodiment of the invention, the target gene may be a fatty acid derivative gene, for example, the target gene is acyl-CoA synthase, thioesterase, ester synthase, alcohol acyltransferase, alcohol dehydrogenase, It may be selected from the group consisting of acyl-CoA reductase and fat-alcohol-forming acyl-CoA reductase.

Upon introduction of the target gene, fatty acid derivatives are expressed to increase the fatty acid content of the seed, thereby obtaining a large amount of vegetable oil or vegetable wax from the seed, which can be used in cosmetics or medicine.

The present invention also provides a method for expressing a target gene in plant seeds by transforming a plant with the recombinant plant expression vector.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration or tissue culture. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Calcium / polyethylene glycol methods for protoplasts (Krens, FA et al. 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) , Agrobacterium tumerfaciens (DNA or RNA-coated) particle bombardment (Klein TM et al., 1987, Nature 327, 70), invasion of plants or transformation of mature pollen or vesicles Appropriately selected from infection with (incomplete) virus (EP 0 301 316) in mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery. Especially preferred is the use of the so-called binary vector technology as described in EP A 120 516 and US Pat. No. 4,940,838.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cells may be cultured cells, cultured tissues, cultured organs or whole plants, preferably cultured cells, cultured tissues or cultured organs and more preferably any form of cultured cells.

"Plant tissue" refers to a tissue of differentiated or undifferentiated plant, including but not limited to roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culture, protoplasts, shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture.

In addition, the present invention comprises the steps of transforming plant cells with a recombinant plant expression vector according to the present invention; And

It provides a method for producing a transformed plant comprising the step of regenerating the transformed plant from the transformed plant cells.

The method of the present invention comprises transforming plant cells with a recombinant plant expression vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens. The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.

The present invention also provides a transgenic plant produced by the method for producing a transgenic plant according to the present invention. The plant is a food crop selected from the group consisting of rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and sorghum; Vegetable crops selected from the group consisting of Arabidopsis, Chinese cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, and carrot; Special crops selected from the group consisting of ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops selected from the group consisting of lysis, redclover, orchardgrass, alphaalpha, tolsquescue and perennial lygragrass.

The transgenic plant according to the invention can be used for oil production. Rape seed oil may be used as a biodiesel raw material, and may also be used as an edible oil and a cosmetic raw material.

The present invention provides a promoter for inducing the expression of genes specifically in plant seeds. The promoter having the plant seed specificity of the present invention can be used for the purpose of specifically expressing the protein of interest in the plant seed, and can not only contain an excessive amount of the useful protein having a medicinal use or physiological activity in the plant seed, as well as fatty acids. Improved plant varieties can be used to industrially use seed oil and improve the productivity of seed plants, including rapeseed.

1 shows seed-specific expression of 12S globulin gene by Northern blot hybridization.
Figure 2 shows the result of separating the promoter region of the 12S globulin gene using the Genome Walking method.
Figure 3 shows the promoter region sequence and Motif analysis of the 12S globulin gene.
Figure 4 shows the preparation of the carrier for deletion analysis according to the Motif position for optimization of the promoter region of the 12S globulin gene.
Figure 5 shows the deletion analysis functional assay of the site of the promoter of the 12S globulin gene in Arabidopsis transformants.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example 1: 12S globulin  Specific expression only in seeds of genes

Total RNA was isolated from various tissues (leaves, stems, roots, petals, and immature seeds) of the Korean cultivars. Subtractive cDNA library was prepared using Youngsan rapeseed seed RNA as a driver and Youngsan rapeseed leaf RNA as a tester, and then a gene group expressed in seeds was obtained by using a self-made rapeseed 300K DNA chip. Based on the genetic information of the 12S globulin gene, which is the most expressed, primers (BnLS484_S: 5'-CAGTGCTGCAACGAGCTCCACCAGGAA-3 ', BnLS484_AS: 5'-AGGAGGGTCCAGGCATGGTCTTCTGGA-3' were prepared to amplify 226 bp gene product obtained by PCR. As a result of sequencing with the pGEM T-easy vector, it was confirmed that it is part of the 12S globulin gene Northern Blot hybridization by a generally known method using a part of the obtained 12S globulin gene as a probe, only from seed It can be seen that it is specifically expressed (Fig. 1) .For reference, Northern blot hybridization by the same method using the 18S ribosomal gene, which is an essential gene present in all tissues, was used as a probe. The total RNA used was well extracted because the expression levels were almost the same. Yongdoen total RNA transfer was found that it was almost the same amount.

Example 2: 12S globulin  Confirmation of nucleotide sequence of promoter region and Motif  analysis

The promoter region of the 12S globulin gene was isolated using the Genome Walking method (GenlonWalker Universal Kit) (Fig. 2), and the sequence of about 1.5 kb in size was transferred to the pCR2.1-topo vector and sequenced. The sequence was confirmed, and Motif of the Cis-acting element was analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) (FIG. 3).

Example 3: Motif  12S according to analysis globulin  For promoter optimization of genes deletion analysis  for Plant transformation  Carrier Fabrication

8 promoter regions in which Motif was removed one by one in the 5 'direction for optimization of the promoter region of the 12S globulin gene (1.5kb, 1.0kb, 0.75kb, 0.6kb, 0.45kb, 0.3kb, 0.2kb, 0.1kb (A of FIG. 4), and based on this, the promoter region of the 12S globulin gene was amplified by PCR, and 8 kinds of plant transformation carriers were prepared to be located in front of the GUS gene using the GateWay vector system (FIG. 4). B).

Example 4: 12S globulin  Of the promoter region of the gene deletion analysis   Function test

In order to investigate the optimization of the promoter region of the rapeseed-derived 12S globulin gene, as shown in FIG. 4, E. coli transformants were produced and transformed into Arabidopsis. As a result, GUS was found in all tissues at 1.5 kb size (A). In the 0.45 kb (B) promoter, GUS staining was observed only in the seed, while in the 0.35 kb (C) promoter, GUS staining was observed, and the 0.2 kb (D) promoter was observed. It was observed that GUS was not stained at all sites (FIG. 5).

Attach an electronic file to a sequence list

Claims (9)

A promoter of 12S globulin gene having the nucleotide sequence of SEQ ID NO: 1. A recombinant plant expression vector comprising the promoter according to claim 1. The recombinant plant expression vector of claim 2, wherein the recombinant plant expression vector is prepared by operably linking a target gene downstream of the promoter. delete delete delete delete delete delete

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