KR101383376B1 - OsTCP6 promoter derived from Oryza sativa and use thereof - Google Patents

Abstract

The present invention is rice (Oryza sativa ) OsTCP6 promoter gene for the expression of a specific protein of interest in seedling meristems, in particular in young ductal tissues of the roots and stems, an expression vector comprising the gene, a transformant transformed with the expression vector, the transformant It relates to a method for preparing and a method of inducing expression of a target protein using the same.
The transformant prepared using the promoter according to the present invention and a recombinant vector comprising the same can be site-specifically expressed in the ductal meristem of rice seedlings. Therefore, it can be usefully used for the production of specific substances, dormancy, germination and growth.

Description

OsTCP6 promoter derived from Oryza sativa and use approximately}

The present invention is rice (Oryza sativa ) -derived OsTCP6 promoter and its use. More specifically, OsTCP6 promoter for the expression of the target protein specific in rice mitotic tissue, more preferably rice seedling meristem, still more preferably seedling ductal meristem, expression vector comprising the same, transformed with the expression vector To a transformant, a method for producing the transformant, and a method of inducing expression of a target protein using the transformant.

Rice is one of the world's most important food crops, and has been steadily increasing its yield over the past two decades. However, the current population growth trend means that by 2020, more than 70% of the current crop will be produced. However, the agricultural land where rice is cultivated is getting smaller and smaller as the industrialization phenomenon of each country in the world, and the new genetic resource which is the subject of breeding is depleted and the introduction of the foreign useful gene is required. Fortunately, the development of molecular biology makes it possible to isolate and manipulate foreign useful genes, transforming these useful genes into many plant cells, including rice, to obtain transformants incorporating new genes. Expression studies, of course, has become a good material for breeding. The production of new varieties using this transformation will not only lead to a high value-added industry in the coming 21st century, but will also solve some of the food problems that are the biggest problem of humanity.

In the 80's, many transformants were obtained by prototyping with polyethylene glycol (PEG) and electroporation. In the late 90's, the use of gene guns became common. Agrobacterium, which has been widely used in dicotyledonous plants, is also widely used. Other methods include pollen pathway and microinjection method.

Promoters affecting the rice transformation efficiency was cloned from CaMV (Cauliflower mosaic virus), rice, corn, soybeans, potatoes, etc., and contributed to the improvement of the transformation efficiency.

The level of marker gene expression has a very important effect on transformation efficiency, and several factors include efficient vector, suitable promoter, location in plant genome, number of copies, 3 'noncoding sequence, and codon frequency. . Promoters determine the pattern of transgenes and two types of promoters have been used, namely, constant and tissue specific. This constitutive promoter independently expresses genes in all tissues. The CaMV35S promoter is a potent, always-on, commonly used transgenic study and has been widely used in rice, wheat, and corn.

Gene expression in target tissues suggests the possibility of crop improvement. Rice is currently used as a model for gene expression and promoter research in monocotyledonous plants, and it is very helpful in understanding the functional interactions between the already identified elements and DNA binding factors. Will be.

Studies on the transformation of rice were carried out in the late 1980s, using the protoplasm, using polyethylene glycol method or electric shock method, and using a total of Agrobacterium genes from Indica. It is reported that a transformant can be obtained. In particular, herbicide resistance (bar) genes, also known as selection genes, have been used most often for transformational research in the 1990s. Since then, new agricultural genes have been developed and manufactured. We use for change over.

For such tissue-specific promoters, US Pat. No. 5,808,034 (Bridges et al., Registered on Sep. 15, 1998) uses genes in the male organs of plants using expression repressors that are specifically promoted by external chemicals. Techniques for regulating fertility have been disclosed by allowing cascades of the cells to occur. In addition, US Pat. No. 6,784,289 (Ouellet et al., Aug. 31, 2004) discloses a technique for controlling expression of a translational regulator that can increase or decrease the expression of a protein involved in a host organism. In addition, promoters specifically expressed in fruit epidermal tissue (KR 813,118) and promoters specifically expressed in plant root tissues (KR 803,390) have been disclosed. In the case of rice, a technique related to a tissue-specific promoter is disclosed in Korean Patent Publication No. 2003-0081377, KR 2001-0085990 and the like.

DME gene is known as a gene showing a tissue-specific expression pattern in meristem of plants. Currently, the function of this gene is not clear, but the function of Arabidopsis DME gene is a gene that is expressed specifically in pistil before modification, especially when it is modified for seed generation. have.

On the other hand, there is no disclosure about a system for expressing useful foreign genes using a promoter specifically expressed in rice, especially seedling meristem. The seedling's meristem is a very important organ that determines plant growth and developmental patterns, and the expression or regulation of useful genes in the meristem may be directly related to the production of specific substances, dormancy, germination and growth. Therefore, as a direct and suitable technique that can regulate the physiological phenomena involved in growth through the regulation of useful gene activity, there is an urgent need for a technique for regulating gene expression specific to these meristems.

The problem to be solved by the present invention is a promoter that specifically expresses a target protein in the seedlings of rice seedlings, in particular, young ducts of roots and stems, an expression vector comprising the same, a transformant transformed with the expression vector The present invention relates to a method for producing the transformant and a method for inducing expression of a target protein using the same.

In order to achieve the above technical problem, the present invention provides a promoter for target protein specific expression in the meristem of rice seedlings having a nucleotide sequence represented by SEQ ID NO: 1, in particular, young ductal tissues of roots and stems.

The present invention also provides a recombinant expression vector produced by operatively linked to a structural gene encoding the promoter and the target protein.

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

According to the present invention, the transformant may be an Agrobacterium tumefaciens transformant.

In the present invention also transformed plants transformed with the expression vector or the Agrobacterium tumefaciens transformant, preferably rice ( Oryza sativa ).

The present invention also provides a method for producing a plant transformant, comprising the step of transforming a plant with the expression vector to express the target protein in the plant.

The plant is rice ( Oryza sativa ), and preferably, the present invention provides a method for producing a rice transformant specifically expressed in the meristem of rice seedlings, in particular the young ductal tissues of roots and stems, comprising the following steps:

(1) a recombinant expression vector operatively linked by operatively linked a promoter for specific expression of a protein of interest and a structural gene encoding a protein of interest in the ductal meristem of rice seedlings comprising the nucleotide sequence represented by SEQ ID NO: 1; Preparing a;

(2) introducing the expression vector into rice; And

(3) selecting a rice transformant that is specifically expressed in the meristem of rice seedlings by introducing the expression vector.

According to the present invention, the step of introducing the expression vector into the rice is transforming Agrobacterium tumefaciens with the expression vector; And infecting the transformed Agrobacterium tumefaciens with rice.

In the present invention, it is also possible to operate a target gene specific expression promoter and a structural gene encoding a target protein in the meristem of rice seedlings, in particular, young duct tissues of roots and stems, including the nucleotide sequence represented by SEQ ID NO: 1. The present invention provides a method of inducing expression of a target protein in endometrial meristem of rice seedlings by transforming rice with a recombinant expression vector prepared by operatively linked.

The promoter of the present invention has a nucleotide sequence of SEQ ID NO: 1, but a sequence having at least 80% homology with the sequence, preferably at least 90% homology, more preferably at least 95% homology, even more preferably 98 A sequence exhibiting at least% homology, most preferably at least 99% homology, comprising bases having substantially the same or corresponding biological activity. In addition, it is obvious that the promoter having a base sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention if it is a base sequence having substantially the same or corresponding biological activity as a sequence having such homology.

In the present invention, "homology" is intended to indicate a degree of similarity with a wild type base sequence, and includes a base sequence of the present invention and a sequence having the same sequence as the percentage or more as described above. Commercially available computer programs can calculate homology between two or more sequences as a percentage and homology can be calculated for adjacent sequences. Can be.

The promoter of the present invention may express the target protein to the target gene RNA in the endometrial meristem of rice seedlings, and more preferably, the target protein may be specifically expressed in the endometrial meristem of the rice seedlings.

In one embodiment of the present invention, as a result of transforming rice with a recombinant expression vector comprising the OsTCP6 promoter of the present invention, the target protein operably linked to the promoter is a ductal meristem of rice seedlings, in particular the young ducts of root and stem. It was confirmed that it is specifically expressed in meristem.

The site specific promoters of the present invention can act as promoters of conventional recombinant expression vectors.

In the present invention, a recombinant expression vector is a vector capable of expressing a target protein or a target RNA in a host cell, and refers to a gene construct including essential regulatory elements operably linked to express a gene insert. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals and enhancers (promoter) and can be prepared in various ways depending on the purpose. Can be. The expression vector also includes a selection marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, the origin of replication.

The recombinant expression vector of the present invention may contain various structural genes known in the art, and these structural genes are operably linked to various promoters known in the art. In the present invention, a promoter means a DNA sequence that controls the expression of a nucleic acid sequence operably linked in a specific host cell, and operably linked means that one nucleic acid fragment is combined with another nucleic acid fragment to function thereof. Or expression is affected by another nucleic acid fragment.

Also, the structural gene may be selected from the group consisting of an antibiotic resistance gene, a herbicide resistance gene, a virus resistance gene, an insect resistance gene, a metabolism-related gene, a luminescent gene, a GFP (green fluorescence protein, AY598428) gene, a GUS (β-glucuronidase, AF485783) (? -galactosidase, AAB49721) gene, and the like.

In one embodiment of the invention, the recombinant expression vector was constructed by operably linking the reporter gene GUS to the pBGWFS7 vector for expression of the promoter using a gateway system.

The present invention also provides a transformant transformed with a recombinant expression vector comprising the promoter of the present invention.

In the present invention, transformation with the recombinant expression vector of the present invention may be performed by transformation techniques known to those skilled in the art. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion PEG-mediated fusion, microinjection, liposome-mediated method, In planta transformation, Vacuum infiltration method, floral meristem dipping method) or Agrobacteria spraying method (Agrobacteria spraying method) may be used, but is not limited thereto.

In addition, the transformant is Escherichia coli ), Bacillus subtilis ( Bacillus subtilis), Streptomyces (Streptomyces), Pseudomonas (Pseudomonas), Proteus Mira Billy's (Proteus mirabilis ), Staphylococcus and Agrobacterium tumefaciens ( Agrobacterium) tumefaciens ) is possible, but is not limited thereto.

In addition, the transformant may be a plant or a plant cell, but is not limited thereto. Plants include whole plants, parts of plants, callus, plant tissues, plant cells and plant seeds. Plants to which the method according to the present invention can be applied include monocotyledonous plants or dicotyledonous plants, preferably monocotyledonous plants, for example, rice, wheat, barley or corn, particularly rice.

In one embodiment of the present invention, the recombinant vector was transformed into Agrobacterium tumefaciens, inoculated in rice to produce a transformant.

In addition, the present invention is prepared by operatively linked (operationally linked) the target protein specific expression promoter and the structural gene encoding the target protein in the ductal meristem of rice seedlings comprising the nucleotide sequence represented by SEQ ID NO: 1 The present invention relates to a method of inducing expression of a target protein in rice seedling meristem by transforming rice with the recombinant expression vector.

The transformant prepared using the promoter according to the present invention and a recombinant vector comprising the same can be site-specifically expressed in the meristem of rice seedlings, especially in young ductal meristem of roots and stems. Therefore, it can be usefully used for the production of specific substances, dormancy, germination and growth.

Figure 1 shows the cis-acting element found in the OsTCP6 promoter.
Figure 2 shows a schematic diagram of the expression vector containing the OsTCP6 promoter for transforming rice prepared through the gateway system, a schematic diagram using GUS as an example of the target protein.
Figure 3 shows the results of genomic PCR analysis of OsTCP6-GUS transformed rice according to the present invention. Here, WT1 and WT2 represent Dongjin rice as a control.
Figure 4 shows the results of GUS staining analysis of transgenic rice in which the OsTCP6 promoter is inserted. Where A) is seedling 5 days after germination, (B) is cotyledon (dyedyne), (C) is side root (dynedyy), (D) is mature seed, (E) is seed derived Callus, (F) is circle root (root, duct dyeing), (G) auxinized seedlings, VB is vascular bundle, E is embryo, C is callus, Col is Coleoptile, LT is leaf apical, and M represents a meristemic region.

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 into various other 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 Cloning and Sequence Analysis of OsTCP6 Promoter Region by PCR of Genomic DNA

First, in order to separate the promoter region of the OsTCP6 gene, base sequence information of 2,039 bp of the base sequence was extracted from the rice genome database, and a primer for amplifying fragments of the promoter region was designed to forward primer OsTCP6P-F: 5 '. Takara's Primestar polymerase was synthesized by synthesizing the genomic DNA of Dongjin rice by synthesizing -CACCCCTTGTGTACCATTAAGATTAATCG-3 '(SEQ ID NO: 2) and reverse primer (OsTCP6P-R: 5'-CAATTAGGCCATGGTATATATACACAG-3' (SEQ ID NO: 3). And amplified the gene using the Applied Biosystem 9700. The PCR reaction conditions were denatured at 95 ° C. for 10 minutes, followed by 94 ° C. for 1 minute, 56 ° C. for 1 minute, and 72 ° C. for 2 minutes. After repeating, the mixture was further treated for 10 minutes at 72 ° C. After the PCR reaction, the amplified gene was electrophoresed in 1% agarose, and the band estimated by OsTCP6 promoter was removed. The cut was purified using a QIAquick gel extraction kit and used for cloning.

The PCR product was cloned into a gateway cloning vector (pENTER / D-TOPO, Invitrogen) and the entire nucleotide sequence was determined and registered in GenBank (JN176138; published on July 31, 2012). An OsTCP6 promoter gene having the indicated nucleotide sequence was obtained.

In order to identify the reaction regulators present in the OsTCP6 promoter, PLACE (Plant Cis-acting Elements database: http://www.dna.affrc.go.jp/PLACE/) and PlantCARE (http: //bioinformatics.psb. Analysis of gene expression motifs using the ugent.be/webtools/plantcare/html/) database program revealed meristem expression motifs (CAT box) and two auxin-reactive motifs (AuxRR, ARFAT) and ABA (ABRE). Contains various hormonal response motifs such as ethylene (ERE), GA (P box), and light-responsive motifs (I box, GATT, Chs unit, GATA) and motifs related to seed expression (RY repeat, O2 site) It was confirmed that a large number exist. The results are shown collectively in FIG. That is, Figure 1 shows the cis-acting element (cis-acting element) found in the OsTCP6 promoter.

Example 2 Preparation of Vector for Rice Transformation

The isolated OsTCP6 promoter gene was introduced into a pBGWFS7 vector (PSB, Plant System Biology) as a target vector. Then, the genes introduced by colony PCR and sequencing were confirmed.

Figure 2 shows a schematic diagram of the expression vector of the OsTCP6 promoter for rice transformation prepared through a gateway system.

Freeze and thaw was repeated 2-3 times to transform the finished vector into Agrobacterium tumefaciens (LBA4404), and then transformed by heat shock at 37 ° C. Colonies were confirmed by overnight incubation in YEP medium (Yeast 10g, NaCl 5g, peptone 10g, Agar 15g / 1L).

Colonies confirmed to be transformed by colony PCR were prepared using AB medium (AB buffer (K ₂ HPO ₄ 60 g, NaH ₂ PO ₄ 20 g / 1 L), AB Salts (NH ₄ Cl 60 g, MgSO ₄ · 7H) for transformation into rice. ₂ O 6g, were cultured in _{KCl 3g, CaCl 2 · 2H 2} O 0.265g, FeSO 4 · 7H 2 O 50mg / 1L), Glucose 5g / 1L).

Example 3 Transformation of Rice Using Agrobacterium

Agrobacterium-based rice transformation was performed by washing the seeds in lacx to induce the callus of rice in 2N6 medium, then drying it appropriately, and 2 mg / D hormone was added to N6 medium (Duchefa vitamin containing medium) 2 mg / L was added to the medium added. After induction of callus by cancer culture at 27 ° C. for 3-4 weeks, embryogenic callus was sub-cultured in 2N6 fresh medium.

Agrobacterium transformed genes containing the transformed genes were cultured in AB liquid medium and cocultured with embryogenic callus for 20 minutes and then cultured for 3 days. Add cefotaxime to sterile water and wash until Agrobacterium is completely removed, and then again in 2N6 medium for 3 weeks in medium containing 6 μg / L of cytotactile and ppt (phosphinothricin, phosphinothricin) Cancer culture. Callus, which survived browning in 2N6 medium containing cephataxim and ppt, was transferred to 2N6 medium containing cetactac and ppt, and cultured for 2 weeks. To induce shooting in the callus, transfer to MSR medium containing cytotactile and ppt (add maltose and sorbitol to Duchefa MS medium) and incubate for 4 weeks. Was carried out.

Genomic DNA was isolated from seedlings of transformed rice and PCR analyzed to confirm that the introduced vector was inserted. As a primer for amplifying the fragment, a forward primer (sense primer Os02g51280P-5: 5'-GAC CCA CCC ACA ACG AGA GGG AGG-3'SEQ ID NO: 4) and a reverse primer (antisense primer EGFP-R: 5'-GGTCTTGTAGTTGCCGTCGTCC- 3 ′ SEQ ID NO: 5) was synthesized.

Figure 3 shows the results of genomic PCR analysis of transformed rice. Here, WT1 and WT2 represent Dongjin rice as a control.

Example 4 Analysis by GUS Protein Activity Staining of Transgenic Rice for Tissue Expression Assay of OsTCP6 Promoter

Transgenic rice strains with confirmed OsTCP6 promoter insertion were grown in MS medium containing PPt (6 μg / L) for 1-5 days, and the seedlings were GUS stained (1 mM X-glu, 0.5 mM ferrocyanide, 0.5 mM). ferricyanide, 10 mM EDTA, 50 mM Na phosphate, 0.1% Triton X-100) and staining within 24 hours, GUS expression was observed in the base of the young seedlings, cotyledon and young leaf apex. In addition, the treatment of auxin (2,4-dichlorophenoxy acetic acid), a hormone that promotes growth and cell division, significantly increased GUS expression in the stems and roots of seedlings. GUS stained samples were treated with tissue clearing solution (Cloral hyrate 500 g, glycerol 62.5 ml. H ₂ 0 125 ml) for 1 day for detailed analysis of GUS staining locations of roots and stems, followed by microscope (Carl Zeiss DE / Axio imager M1). ) And the image analysis program (AxioVision Imaging System). The results demonstrate that the OsTCP6 promoter specifically induces GUS expression in the cotyledons of cotyledons and roots, especially at the beginning of young root origin. In addition, it was confirmed that the OsTCP6 promoter has a characteristic of inducing GUS expression in embryos and seed-derived callus of mature seeds.

Through this, OsTCP6 promoter was able to express the target gene in young ductal meristem mainly in rice, which is a typical monocotyledonous plant, and it was able to induce the expression more strongly by controlling auxin concentration, a hormone that promotes cell division.

Figure 4 shows the results of GUS protein staining analysis of transgenic rice in which the OsTCP6 promoter is inserted. Here, (A) is seedling 5 days after germination, (B) is cotyledon (dyedyne), (C) side root (dyedyne), (D) mature seed, (E) seed Derived callus, (F) is circle root (root, duct dyeing), (G) auxinized seedlings, VB is vascular bundle, E is embryo, C is callus, Col Is a coleoptile, LT is a leaf apex and M represents a meristemic region.

The transformant prepared by using the promoter according to the present invention and a recombinant vector comprising the same can positionally express a target protein in endometrial meristem of rice seedling seedlings, particularly young ductal meristem of root and stem. have. Therefore, it can be usefully used for the production of specific substances, dormancy, germination and growth.

Attach an electronic file to a sequence list

Claims (14)

A promoter for specific expression of a protein of interest in meristem of rice seedlings having a nucleotide sequence represented by SEQ ID NO: 1. The promoter of claim 1, wherein the meristem is a vascular tissue of roots and stems. A recombinant expression vector produced by operatively linked the promoter of claim 1 or 2 and a structural gene encoding a protein of interest. A medium for transformation of plants transformed with the vector of claim 3. The method of claim 4, wherein the medium for transformation of the plant is Agrobacterium tumefaciens, the medium for transformation of the plant, characterized in that. Transgenic plant transformed with the vector according to claim 3. The method of claim 6, wherein the transgenic plant is Oryza sativa ), characterized in that the transgenic plant. A method for producing a plant transformant comprising transforming a plant with the vector of claim 3 to express the target protein in the plant. The method of claim 8, wherein the plant is Oryza sativa ). 10. The method of claim 9,
A method for preparing a rice transformant specifically expressed in endometrial meristem of rice seedlings comprising the following steps:
(1) Recombinant expression by operably linking a promoter-specific expression promoter and structural gene encoding a protein of interest in the ductal meristem of rice seedlings comprising the nucleotide sequence represented by SEQ ID NO: 1 Preparing a vector;
(2) introducing the expression vector into rice; And
(3) selecting a rice transformant that is specifically expressed in the meristem of rice seedlings by introducing the expression vector. The method of claim 10, wherein the introducing of the expression vector into the rice comprises: transforming Agrobacterium tumefaciens with the expression vector; And infecting the transformed Agrobacterium tumefaciens with rice. With a recombinant expression vector produced by operably linked (operationally linked) a target protein specific expression promoter and structural gene encoding the target protein in the ductal meristem of rice seedlings comprising the nucleotide sequence represented by SEQ ID NO: 1 A method of transforming rice to induce expression of a target protein in the ductal meristem of rice seedlings. Transgenic plant transformed with a medium for transformation of the plant according to claim 4. The transformed plant according to claim 13, wherein the transformed plant is rice ( Oryza sativa ).

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