KR101965971B1 - Phosphate starvation inducible promoter and use thereof - Google Patents

Abstract

The present invention relates to a promoter that preferentially induces a foreign gene to a root in a phosphate-deficient environment. The promoter of the present invention, capable of preferentially expressing a foreign gene in a root in a phosphate-deficient environment, can be used to improve useful traits of roots of major cereals or monocotyledons including Oryza sativa in a phosphate-deficient environment by regulating the gene expression necessary for improvement of root traits in the case of phosphates deficiency. The present invention provides a composition for inducing root-first expression of a foreign gene in the phosphate-deficient environment, comprising a promoter consisting of a nucleotide sequence of SEQ ID NO: 1 and a vector comprising a foreign gene operatively linked to the promoter.

Description

Phosphate Deficiency Inducible Promoter and Its Use {Phosphate starvation inducible promoter and use thereof}

The present invention relates to a novel promoter capable of inducing a foreign gene into the root in a phosphate-deficient environment and its use.

Rice (Oryza sativa) is one of the most important food crops in the world. It has been steadily increasing its yields over the past two decades, but by 2020 it will have to produce more than 70% have. 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 has enabled the isolation and manipulation of exogenous useful genes, transforming useful genes into many plant cells, including rice, to obtain transformants with new genes, Research has become a good source of breeding as well. The production of new varieties using this transformation will not only become a high value - added industry in the coming 21st century, but it will also solve some of the food problems that are highlighted as the greatest problems of mankind.

Many transgenic plants have been obtained by using polyethylene glycol (PEG) and electroporation in the protoplasts of the 1980s as a known transformation method. In the late 1990s, Agrobacterium, which has been widely used in the art, is also widely used. Other methods include pollen pathway and microinjection method.

The promoter is a genomic region connected to the upper side of the gene, and plays a role of regulating transcription of a structural gene linked to the promoter to mRNA. Since the proteins necessary for basic metabolism must maintain a certain concentration in the cells, the promoter linked to these genes is always activated by the action of a common transcription factor. Conversely, proteins that do not have a role in normal times and that require function only under specific circumstances may be activated by binding environmental transcription factors or specific transcription factors activated by external stimuli.

That is, a promoter can achieve the purpose of transformation by limiting the expression of a foreign gene to a whole body or a specific tissue of a plant as necessary, and may include the following promoters depending on function.

First, systemic expression-inducible promoters can be mentioned. As a plant systemic expression-inducing promoter, a promoter of 35S RNA gene of cauliflower mosaic virus (CaMV) is used as a typical promoter for dicotyledonous plants. Actin and maize ubiquitin gene promoters have been mainly used as promoters for the expression of the whole plants in rice plants and rice plants. Recently, a promoter of the rice cytochrome C gene (OsOc1) has been developed by domestic researchers, (Korean Patent No. 10-0429335). They are already inherent in inducing the expression of antibiotics, herbicide resistance genes and reporter genes used as selection markers in the plant transgenic carrier. From a research point of view, Promoters considered. However, overexpression of certain genes linked to these promoters often results in the side effects of transgenic plants not being germinated or being dwarfed due to the toxic effects of large amounts of proteins that are unnecessary for biological metabolism. As a representative example, it was observed that the Arabidopsis thaliana overexpressing the environmental stress transcription factor gene DREB1A using the CaMV35S promoter promoted resistance to low temperature and low moisture conditions. However, the growth was inhibited and the dwarfed phenotype of amino acid proline and (Lie et al., Plant Cell 10: 1391-1406, 1998; Gilmour et al., Plant Physiol. 124: 1854-1865, 2000). This side effect can be minimized by using an inducible promoter of the environmental stress-related gene rd29A instead of the CaMV35S promoter (Kasuga et al., Nat. Biotechnol. 17: 287-291, 1999).

Second, seed-specific promoters can be mentioned. As a representative example, the rice glutelin promoter used for the development of golden rice as promoters of major storage protein genes of rice has been widely used to induce seed-specific expression of monocotyledonous plants to date, Promoter of soybean-derived lectin, napin promoter derived from Chinese cabbage, and gamma-tocopherol methyl transferase (gamma-TMT) in the seeds of Arabidopsis thaliana ) Patent application (Korean Patent No. 10-0685520) for inducing seed-specific expression of carrot-derived DC-3 promoter and perilla originated oleosin promoter used in the study to promote vitamin E production by inducing gene expression have. The seed-specific promoters are mainly used for the purpose of accumulating useful proteins and producing beneficial substances in major crops in which seed itself is used as a food, a food, or a raw material for food.

Third, other tissue specific promoters such as leaves may be mentioned. (RbcS: ribulose bisphosphate carboxylase / oxygenase small subunit) promoter which induces expression of a strong gene only in photosynthetic tissues such as leaves, RolD promoter inducing expression of plant roots derived from Agrobacterium, potato-derived tuber A specific expression-inducible patatin promoter, and a tomato-derived fruit maturation-specific expression-inducing PDS (phytoene synthase) promoter.

Fourth is root specific expression promoter. (IbMADS) and sugar-induced adipose-glucose pyrophosphatase (AGPase) genes were isolated from the sweet potato-derived rice paddy rice, and the peroxidase (prxEa) (Korean Registration Nos. 10-0604186 and 10-0604191), confirming that the promoter induces specific expression in roots and induces root-specific transient expression in carrot and radish.

Most of these promoters are promoters that induce the expression of a foreign gene at a certain level without being affected by specific environmental conditions. Since the pro-amers do not consider the activity change as a promoter according to the survival environment of the plant or the specific stress condition, there is a problem that the expression of the foreign gene can not be manipulated in an environment capable of causing disease in the plant. Accordingly, a stress inducible promoter capable of inducing the expression of a target gene in a specific condition or at a specific time, rather than a promoter that induces expression at all times in the plant tissue, is required.

Most of the systems that induce the expression of specific genes according to these stresses were chemical substances. For example, a method of treating a chemical substance such as steroid dexamethasone, antibiotic tetracycline, copper ion, and IPTG with an inducer has been known (Gatz et al., Plant J. 2: 397-404, 1992 ; Weimann et al., Plant J. 5: 559-569, 1994; Aoyama T. and Chua NH, Plant J. 11: 605-612, 1997). However, there is a problem that the price of the inductive compound used in these systems is extremely high, the toxic effect of the chemical itself is generated, and it is not applicable to all plants.

For the induction of foreign genes in a stress environment by genetic manipulation, the following promoters are known for example. Korean Patent No. 10-0781059 discloses an inducible promoter which is activated by environmental stress and a method of obtaining a transgenic plant which specifically produces a target protein in the guinea pig cells using the promoter. Korean Patent Laid-Open No. 10-2010-0107263 also discloses that a Wai gene promoter or an ABA-induced promoter derived from rice is derived from a rice plant under salinity stress and drought stress conditions. Korean Patent No. 10-1131770 discloses that a dry stress-inducible ADF (actin depolymerization factor) promoter or AWPM promoter derived from rice specifically induces a foreign gene in drying stress.

On the other hand, phosphoric acid is one of the essential nutrients required by plants and plays an important role in metabolism in plant cells such as energy transfer, signal transduction, biosynthesis of metabolites, photosynthesis and respiration, It is a nutrient element that plants most need together with nitrogen as an ingredient. The large amount of phosphoric acid required for the growth of plants is supplied mainly from plants in the form of fertilizers, but most of the phosphoric acid is immobilized in the soil in combination with organic or metal ions that are not available to plants. Therefore, the available amount of available phosphoric acid in actual plants is very small in soil. Particularly, in the case of acidic soil, the phosphoric acid is further immobilized by excessively accumulated aluminum ions, thereby lowering the effectiveness of the soil. Therefore, new varieties that can resist these phosphorus deficient environments are needed.

Considering the above technical situation, a new promoter which can control the place of gene expression more precisely according to the intention of the developer, for example, a gene which should be expressed only in a specific environment (for example, drought, salt stress, nutrient deficiency, Lt; RTI ID = 0.0 > promoter. ≪ / RTI >

Korean Registered Patent No. 10-0429335 2004.06.16. Korean Registered Patent No. 10-0685520 2007.02.14. Korean Registered Patent No. 10-0604186 2006.07.18. Korean Registered Patent No. 10-0604191 2006.07.18. Korean Registered Patent No. 10-0781059 2006.12.01. Korean Patent Publication No. 10-2010-0107263 2010.10.05. Korean Registered Patent No. 10-1131770 2012.03.23.

The present invention provides a promoter for inducing root-first expression of a foreign gene capable of expressing foreign genes in a phosphate-deficient environment.

The present invention provides an expression vector comprising a promoter for inducing root-first expression of a foreign gene capable of expressing a foreign gene at a root priority in a phosphate-deficient environment and a foreign gene operably linked thereto.

The present invention provides a composition for inducing root-first expression of a foreign gene, comprising a promoter for inducing root-first expression of a foreign gene and a vector comprising an operably linked foreign gene in a phosphate-deficient environment.

The present invention relates to a method for inducing root-first expression of a foreign gene, comprising a transgenic cell transformed with a vector comprising a promoter for inducing root-first expression of a foreign gene and a foreign gene operably linked thereto in a phosphate-deficient environment ≪ / RTI >

The present invention provides a transgenic monocotyledonous plant which is transformed with the above composition for inducing root induction expression and preferentially expresses a foreign gene in the root of a terminal plant in a phosphate-deficient environment.

The present invention relates to a method for producing a foreign gene in a phosphate-deficient environment, which comprises transforming a monocot plant with a vector comprising a promoter for inducing root-first expression of the foreign gene and a foreign gene operably linked thereto in a phosphate- Which is expressed primarily in the root, is provided.

The present invention relates to a method for producing a transgenic monocotyledonous plant in which a foreign gene is preferentially expressed in a root in a phosphate-deficient environment,

(a) a composition for inducing root-first expression of a foreign gene in a phosphate-deficient environment, comprising a vector comprising a promoter for inducing root-first expression of a foreign gene and a foreign gene operably linked thereto in a phosphate-deficient environment Producing;

(b) introducing the composition into a terminal plant; And

(c) selecting the transgenic monocotyledonous plants to which the composition is introduced such that the foreign gene is expressed primarily in a phosphate-deficient environment.

In order to achieve the above object, the present inventors have discovered that promoters capable of expressing a foreign gene in a tissue or organ-specific manner by external stimulation of a plant, thereby locating the promoter specific to a specific tissue or organ of the plant, Expression or inhibition of the < / RTI >

For this purpose, a study was conducted on promoters capable of inducing foreign genes in a phosphate-deficient environment. As a result, it was confirmed that the promoter of LOC_Os02g44654 in rice induces the expression of foreign genes preferentially in the root at the time of phosphate deficiency.

Thus, the present invention has been accomplished by securing the promoter, preparing an expression vector containing the promoter, and introducing the expression vector into a plant to confirm that the transformant expressing the reporter gene is preferentially expressed in a phosphate-deficient environment.

More specifically, the present inventors referred to published RNA-RNA data to isolate 774 genes whose expression level is increased in the root when 21 days of phosphorylation stress is given. The LOC_Os02g44654 gene of the above genes showed a marked increase in expression at the root only when the phosphate deficiency was detected. To verify this, the expression of phosphoric acid was gradually increased at 7 days and 21 days using real-time PCR technique Respectively.

Based on this, the region containing the promoter region from -1301 to +1110 and the part of the first exon (total 2,411 bp) of LOC_Os02g44654 was isolated by genomic DNA PCR, and pGEM T -Easy vector and the base sequence was decoded.

The thus isolated promoter was inserted into the pGA3519 vector to prepare a transformed plant. By inspecting the GUS reaction after 21 days of phosphorus deficiency stress in the transgenic plants, the promoter induces root-first gene expression in the absence of phosphoric acid, thereby completing the present invention.

The present invention provides a promoter for inducing root-first expression of a foreign gene in a phosphate-deficient environment. In the phosphate-deficient environment according to the present invention, the promoter for inducing the primary preferential expression of the foreign gene includes the promoter region of LOC_Os02g44654 of rice, and more preferably the nucleotide sequence shown in SEQ ID NO: 1.

The promoter according to the present invention imparts resistance to the stress environment of phosphate deficiency by inducing root-first expression of the foreign gene in a phosphate-deficient environment, for example, in an environment lacking phosphorus in the soil.

Promoters are essential elements for regulating the environmental, temporal, and systemic expression of genes. Promoters according to the present invention can preferentially induce the expression of exogenous utility genes in plant roots in a phosphate-deficient environment. Preferably at the distal end of the root, more preferably in the kidney of the root.

That is, the promoter of the present invention is a promoter of LOC_Os02g44654 of rice, and is a phosphorylation-inducible root-preferential expression promoter (hereinafter referred to as 'the promoter of the present invention') which induces the expression of a foreign gene preferentially to the root at the time of phosphate deficiency.

In the present invention, " preferential " means a preferential expression increase in a part of a plant including all of the high specificity and specificity by the promoter according to the present invention. For example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 200% or more, 5 or more times or more increase in expression compared to a plant lacking the promoter according to the present invention it means.

The promoter of the present invention is, for example, a promoter region from -1301 to +1110 of LOC_Os02g44654. The promoter shown in SEQ ID NO: 1 of the present invention and a part of the gene region are located in front of LOC_Os02g44654 on the public rice genome sequence, and a part of the gene, and some of the nucleotide sequences shown in SEQ ID NO: 1 are substituted, inserted or deleted , It can be included in the scope of the present invention if homology with the promoter of LOC_Os02g44654 on the published rice genome sequence and the sequence located in some gene regions are maintained and exhibit substantially equivalent effects. That is, it may be a fragment or a fragment of a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, which may contain the " promoter of the present invention " and " functionally equivalent fragment ", and exhibits substantially equivalent effect to the promoter. These nucleic acid fragments are 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, 99% or more of sequence homology. Such a nucleic acid fragment can be easily prepared by molecular biological methods well known in the art. Preferably, it comprises the nucleotide sequence of SEQ ID NO: 1.

The invention also provides an expression vector comprising said promoter and an operably linked foreign gene. The above promoter is a promoter for inducing root-first expression of a foreign gene capable of expressing foreign genes in a phosphate-deficient environment as described above.

The term " vector " refers to a DNA fragment (s), nucleic acid molecule, which transfers into a cell, which can be cloned and independently reprogrammed in host cells. &Quot; Expression vector " means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may generally be derived from a plasmid or viral DNA, or may contain both elements. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that replicate in eukaryotic and / or prokaryotic cells and those that remain as episomes or that are integrated into the host cell genome.

Such a vector may be any of those which can introduce the promoter of the present invention, but is preferably a pCAMBIA family, a pGA family, a pGWB family such as pGA3519, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14 Ti-plasmid, and vectors derived therefrom. These vectors contain the promoter and the foreign gene of the present invention and have a remarkable effect on the expression of the foreign gene preferentially in the root at the time of deficiency of phosphoric acid.

The expression vector of the present invention includes a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a functionally equivalent fragment thereof.

The expression vector of the present invention allows expression of a foreign gene for the target protein in the 3 'direction. Preferably, the expression vector may comprise a useful foreign gene that is preferentially expressed in the root upon a phosphate deficiency.

There are no restrictions on the types of these foreign genes. However, in order to grow plants, the absorption of nutrients and water is important. Roots play a major role, and productivity can be increased by improving nutrient absorption ability through roots. Therefore, it is preferable that the foreign gene is related to the above-mentioned characteristics for improving the nutrient absorption ability and the root mentioned above when the phosphate deficiency occurs. For example, Phosphate transporter 6, WRKY76, etc. of rice may be introduced as a gene related to root development in the case of phosphoric acid deficiency. In addition, OsPT4 genes involved in nutrient absorption can be introduced, and examples of such foreign genes are not limited to the types of genes that can be introduced.

The above expression vector may be one in which the promoter of the present invention is introduced into a conventional vector, and the expression vector may be prepared by introducing the promoter of the present invention into such a manner as will be readily apparent to those skilled in the art. It is possible.

The present invention also provides a transformed cell transformed with said vector.

Such recombinant vectors 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 bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; And plant 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. &Quot; 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.

The present invention also provides a composition for inducing root-first expression in a phosphate-deficient environment of a foreign gene comprising the expression vector or the transformed cell. A composition for inducing root-priority expression in a phosphate-deficient environment can preferentially induce the expression of a foreign gene in a root when a foreign gene is introduced into a terminal plant and the transformed terminal plant is exposed to a phosphate-deficient environment. In the present invention, the monocotyledonous plant includes, without limitation, all of the monocotyledonous plants including rice ( Oryza sativa ) according to one embodiment of the present invention. In the present invention, induction of the primary preferential expression in the absence of phosphoric acid means preferential expression in the root in the condition of phosphorylation of the terminal plant.

The present invention also provides a transgenic plant transformed with the vector, the transfected cell or a composition for inducing root-first expression comprising the transfected cell. The terminal plant is preferably rice and the terminal plant is rice. These transgenic monocotyledonous plants exhibit the preferential expression of the introduced foreign gene in the root of the terminal plant when it is deficient in phosphoric acid.

65%, 70%, 75%, 80% or more of the variants of the nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 " and " functionally equivalent transgenic plant of the present invention " , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology As a terminal plant, it is a transgenic terminal plant having a characteristic that the promoter effect is substantially equivalent in the terminal plant.

Transformation of a plant of the present invention can be carried out by any method known in the art for transferring DNA to a plant. Such transformation methods do not necessarily have a regeneration and / or tissue culture period. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. 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 mature pollen of various plant elements (DNA or RNA-coated) And infection by (non-integrative) viruses in Agrobacterium tumefaciens mediated gene transfer (EP 0301316). Such mutants can be easily produced by molecular biological methods well known in the art.

The present invention also relates to a method for the isolation of a foreign gene in a phosphate-deficient environment, comprising the step of transforming the monocotyledonous plant into a vector comprising a promoter for inducing root-first expression of the foreign gene and a foreign gene operably linked thereto in a phosphate- A method for producing a transgenic monocotyledonous plant which expresses the gene preferentially in the root.

The present invention also relates to a method for producing a transgenic monocotyledonous plant in which a foreign gene is preferentially expressed in a root in a phosphate-deficient environment,

(a) a composition for inducing root-first expression of a foreign gene in a phosphate-deficient environment, comprising a vector comprising a promoter for inducing root-first expression of a foreign gene and a foreign gene operably linked thereto in a phosphate-deficient environment Producing;

(b) introducing the composition into a terminal plant; And

(c) selecting the transgenic monocotyledonous plants to which the composition is introduced such that the foreign gene is expressed primarily in a phosphate-deficient environment.

The promoter of the present invention is a promoter capable of preferentially expressing a foreign gene in a root in a phosphate-deficient environment. It can regulate gene expression necessary for improving the root character in the absence of phosphoric acid, Can be used to improve useful traits of roots.

FIG. 1 is a heatmap showing genes showing preferential expression changes in root at the time of phosphate deficiency and their expression patterns. FIG.
Fig. 2 is a diagram showing the results of RNA-seq data showing specific expression only at the root in the absence of phosphoric acid of LOC_Os02g44654 gene.
FIG. 3 is a diagram showing expression patterns of the LOC_Os02g44654 gene using a real-time PCR technique in order to show expression changes in the root when the phosphate deficiency occurs.
Figure 4 is a diagram of a vector used for the production of a GUS plant containing the promoter of LOC_Os02g44654.
FIG. 5 is a graph showing the result of GUS staining in which the LOC_Os02g44654 promoter in the transgenic plant shows preferential expression in the root at the time of phosphorus deficiency.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following embodiments are only examples for helping understanding of the invention, and thus the scope of the present invention is not limited thereto.

< Example  1> Large-scale selection of rice genes preferentially expressed in roots in the absence of phosphoric acid

To select genes that preferentially express in phosphate-deficient environments, the RNA-seq database NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/) ) With SRA097415 (phosphate depletion treatment for 1 hour, 3 hours, 6 hours, 24 hours, 3 days, 7 days, 21 days, 21 days for 1 hour, 21 days for 6 hours, or 21 days for 24 hours) And a total of 138 samples confirmed by three iterations on the sample).

In order to screen for genes specifically expressed in the root at the time of phosphorylation deficiency, 744 genes were preferentially selected from published RNA-seq data, and gene selection analysis was carried out using the gene for expression in phosphate-deficient roots.

Using the K-means clustering analysis method, a gene group with a high expression level in the roots was isolated based on the 21 days of phosphorus deficiency treatment in the proposed database for gene expression analysis.

This was performed using MeV's K-means clustering analysis software for analyzing transcript data. In the database for analysis of root expression in the absence of phosphate, 744 genes having preferential expression in the root were isolated in the deficient condition.

The results are shown in Fig.

As shown in FIG. 1, the gene exhibiting a stronger expression toward the yellow color is closer to the green color, and the expression is lowered. Since seeds of plants contain many nutrients, the initial expression of phosphate deficiency is less reliable. Therefore, we identified the top 744 genes of the sample treated with phosphate - depleted 21 days after germination 14 days.

< Example  2> Published ARENA  Analysis of Expression of Phosphate Deficiency by Time Data Analysis

Analysis of the expression of LOC_Os02g44654 gene in the roots and stems at the time of phosphate deficiency through analysis of the published RNA-seq database was performed and the results are shown in Fig.

As shown in FIG. 2, the LOC_Os02g44654 gene changes significantly from the root to the yellow under the phosphoric acid-deficient condition, indicating that stress-specific expression of the gene is preferentially induced by the LOC_Os02g44654 promoter.

< Example  3> Analysis of Root Expression Patterns in Phosphate Deficiency Using Real-Time Chain Polymerization

Previous data analysis showed that the expression of LOC_Os02g44654 gene was increased at the root at the time of phosphate deficiency, and based on this, using real-time PCR technique that quantifies the amplification process in real time, the expression of LOC_Os02g44654 gene in root was increased I could confirm. The results are shown in Fig. As can be seen in FIG. 3, the expression was significantly increased in the phosphate-deficient environment, especially when the phosphate-depleted sample was treated for 21 days than the 7-day treated sample.

< Example  4> Promoter inducing root expression in the absence of phosphoric acid Cloning

PCR was performed using the primers prepared to clone the promoter of LOC_Os02g44654 and the genomic DNA of Oryza sativa L. japonica cultivar-group. Template DNA was used at 100 ng and each primer was used at 5 pmol. PCR conditions were 5 min at 95 ° C; Repeating 4 times at 95 캜 for 30 seconds, at 56 캜 for 30 seconds and at 68 캜 for 300 seconds; 30 seconds at 95 ° C, 30 seconds at 62 ° C, and 300 seconds at 68 ° C for 35 times and finally 68 ° C for 10 minutes. The PCR product was electrophoresed to confirm its size and cloned into a pGEM T-EG vector and sequenced. The cloned DNA was digested with Kpn1 and Xba1, followed by ligation with a pGA3519 vector (vector for promoter-GUS cloning) digested with the same restriction enzyme at 14 DEG C for 12 hours. The ligation product was mixed with 200 [mu] l of Top10 E. coli competent cells, transferred to a 1.5 ml tube, and left on ice for 15 minutes. Subsequently, the plate was allowed to stand in a 37 ° C oven for 1 minute, and then 1 ml of LB liquid medium was further placed in the tube and left in a 37 ° C shaking incubator for 3 hours. Then, the cells were plated on tetracycline-resistant LB solid medium and the resulting colonies were awaited for 12 hours and then mini-prepared after cell culture in 1 mL of LB liquid medium. The DNA obtained as miniprep was digested with restriction enzymes Kpn1 and XbaI, and the agarose gel was separated by electrophoresis and the band was confirmed. The cloning DNA thus obtained was subjected to base sequence analysis again to select DNA that did not cause an error. Nucleotide sequencing was performed by capillary sequencing (Macrogen), and primers using NGUS1 and promoter sequences were used for the nucleotide sequence analysis.

Primers for promoter cloning and primers used for base sequence analysis are shown in Tables 1 and 2 below.

Table 1. LOC _ Os02g44654's  Promoter For cloning primer

Table 2. Sequence analysis primer

The sequence analyzed above is shown in SEQ ID NO: 1, and a promoter region from -1301 to +1110 of LOC_Os02g44654 and a region including a part of the first exon were used as a promoter sequence.

< Example  5> Production of transformed cells

The plant expression vector prepared in Example 4 was extracted.

Agrobacterium competent cells (Agrobacterium tumefaciens LB4404) and 2 μg of vector for plant expression were mixed and left on ice for 15 minutes. Subsequently, the substrate was placed in liquid nitrogen for 75 seconds. Then, the substrate was allowed to stand in an oven at 37 ° C for 5 minutes. Then, 1 ml of LB liquid medium was added thereto, followed by incubation in a 28 ° C shaking incubator for 3 hours. Then, the cells were plated on a tetracycline-resistant LB solid medium and incubated for 36 hours. The resulting colonies were cultured in 1 mL of LB liquid medium, followed by mini-preparation, and Kpn1 and Xba1 were used for enzyme cleavage.

The resulting transformed Agrobacterium was used for transgenic rice transformation experiments.

< Example  6> Production of Transgenic Plants

In the N6D solid medium, Dongjinbyeon seeds were grown in a 28 ℃ growth chamber for 7 days to produce calli of rice. The resulting callus was mixed with the cells cultured for 72 hours with Agrobacterium transformed with the plant expression vector obtained in Example 5 and left in a culture medium containing N6D-Acetosyringone at a temperature of 22 占 폚.

The callus contaminated with Agrobacterium was rinsed 5 times with tertiary distilled water. Subsequently hygromycin selection was performed in N6D solid medium (hygromycin 30 mg / L) and subculture (hygromycin 40 mg / L) . Selection was made in the 28 ℃ growth room for 2 weeks each for 4 weeks. The divided callus was transferred to the MSR (hygromycin 40 mg / L) solid medium for regeneration and induced to regenerate in a light condition growth chamber for 4 weeks at 28 ° C. The plants were transferred to MS solid medium, grown in a light condition growth room for 7 days, To grow regenerated plants.

< Example  7> Validation of induction of phosphate deficiency induced by transgenic plants

To identify the stress-specific GUS expression of the regenerated plants, 10 seeds were cultured at 28 ° C for 7 days, and then all the seeds were cultured in Yoshida nutrient solution (1.425 mM NH ₄ NO ₃ , 0.513 mM K ₂ SO ₄ , 0.998 mM CaCl ₂ , 1.643 mM MgSO ₄ , 0.075 μM (NH ₄ ) ₆ Mo ₇ O ₂₄ , 0.25 mM NaSiO ₃ , 0.009 mM MnCl ₂ , 0.019 μM H ₃ BO ₃ , 0.155 μM CuSO ₄ , 0.152 μM ZnSO ₄ , containing EDTA-Fe, and the phosphoric acid sufficient further comprising a 0.323 mM NaH ₂ PO ₄ and deficiency conditions are then grown 21 days under water culture does not contain a NaH ₂ PO ₄₎ was treated with GUS staining. The GUS solution was dissolved in 200 ml of 0.5 M sodium phosphate buffer, 100 ml of 12.5 mM potassium ferricyanide, 100 ml of 12.5 mM potassium ferrocyanide, 20 ml of 0.5 M EDTA, 50 ml of 10% Triton X-100 and 10 ml of DMSO 1 g of X-gluc and 200 ml of methanol were added, and the volume was adjusted to 1 L with distilled water. All plants were treated with 15 ml of GUS staining solution for 24 hours, followed by decolorization at 70% ethanol and 100% ethanol at room temperature.

The results are shown in Fig. Fig. 5A shows the roots of the plants grown for 21 days under sufficient phosphoric acid conditions, and B, C, D and E show the roots of the plants on the 21st day of phosphate deficiency. Figs. 5A and 5B are photographs of roots as a whole, and C, D and E are enlarged photographs of a part of roots.

As shown in FIG. 5, the transgenic plants showed predominant expression of GUS in the root only in the phosphorus deficient condition, and especially in the distal part of the roots, especially in the elongated part of the roots.

As can be seen from the results of FIGS. 2, 3 and 5, it can be confirmed that the promoter of LOC_Os02g44654 is expressed preferentially in the case of phosphoric acid deficiency. That is, it was confirmed from the above analysis that the promoter region of the LOC_Os02g44654 gene induces root-first expression of the foreign gene in the phosphate-deficient environment.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Phosphate starvation inducible promoter and use thereof <130> P17-132-KHU <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 2411 <212> DNA <213> Oryza sativa <220> <221> promoter &Lt; 222 > (1) <400> 1 tagtagccgt atcacattac atgcgcgctg cggcacacaa ttgaagtctt cgagttgatc 60 tgccggtaga tctagctaag ctggagtcac tttacacttg catttgcatc tgcattgaac 120 tgcactgcat tgcattgcag ttgcactccg tctatctagc tagctagcca cgcatatgtg 180 tactgcgtgc agatggatcc ccatttaagc gtctgcgcct ccgccaccac catccccatg 240 gccacccttc aatgcggccg tgcattcaat ggctcgcccc gcggttaggt gcccgtcgat 300 caccgcggct gatcaagtga tcattgcctc attggccact gatccatcca tcgatcggtc 360 ggacgcccaa cggcccgcgc aggcgccaat ttatggtcga ccggctccgt gctcctcgcg 420 gccgagcgac cgaaccggtc accaatcaca aggcaccaca aactatggtg atcaatggat 480 cgactcacca ctcgcaaggc accacaaact ctgatcctga tcaatggatc gatgcagaag 540 tcctcgtcgt aggtagtgtt cccttcaaac tttcaacttt tccatcacat caaatattta 600 gacacatgca tagagcatta aatatagatg aaaaaaccaa ttacacagtt tgcatgtaaa 660 ttgcgagacg aatcttttaa tcctaattac accatgattt gacaatgtag tgctacagta 720 aacatttact aatgacggat taattagact taataaattt gtctcgtagt ttacaggcgg 780 aatctgtaat ttgttttgtt attggtttac atttaatatt ttaaatgtgt attggtatgc 840 gtcaattttt ttttttgcca agacaactaa gcacggcctt agtatatgcg agtgtagcgt 900 acatgcatgt gtgtacgtag gatcggagtt gttgcgtgac ttgcgtcctg ctgctgcatc 960 gttcatgtac actggtacat gcagagagag agagagagag agagagagag agagagagag 1020 agagagagag agagagctcg cgcttggtgg tagcagctag gagtagcagc gtagcaggac 1080 cggtgcgcga cctgcaataa attggcatcc atctcgactg tgctccgacc agttctataa 1140 aaacctcggt ctctccctgc tgcactctgc catcctcatt gcttgctcca tgtggagtca 1200 cttagcgcag tgtagctagc catcaccggg cattctcatc ggatccatac agccctagct 1260 agcagctgta cgtacattac agagtttgtt gccagaggaa aatggaggtg gggacgtggg 1320 cggtggtggt ggcggtggcg gcggcgtaca tgacgtggtt ctggcggatg tcccgggggc 1380 tgagcgggcc gcgtgtgtgg ccggtggtcg gcagcctgcc tgggctggtg cagcacgccg 1440 agaacatgca cgagtggatc gccgccaacc tgcgccgcgc cggcggcacg taccagacgt 1500 gcatcttcgc ggtgcccggg gtggcgcgcc gcggcgggct ggtcaccgtc acctgcgacc 1560 cgcgcaacct ggagcacgtc ctcaagtcgc ggttcgacaa ctaccccaag ggccccttct 1620 ggcacgccgt cttccgcgac ctgctcggcg acggcatctt caactccgac ggcgagacgt 1680 gggtcgcgca gcggaagacg gcggcgctcg agttcaccac gcgcacgctg cggacggcaa 1740 tgtccaggtg ggtctcgcgg tccatccacc accggctgct gcccatcctg gacgacgcgg 1800 ccgccggcaa ggcgcacgtc gacctgcagg acctcctcct ccgcctcacc ttcgacaaca 1860 tctgcggcct ggcgttcggc aaggaccccg agacgctcgc caagggcttg ccggagaacg 1920 ccttcgcctc cgcgttcgac cgcgcgaccg aggccacgct caaccggttc atcttcccgg 1980 agtacctgtg gcgctgcaag aagtggctgg gcctcggcat ggagaccacg ctggccagca 2040 gcgtcgcgca cgtcgaccag tacctcgccg ccgtcatcaa ggcgcgcaag ctcgagctcg 2100 cgggcaacgg caagtgcgac accgtggcga tgcatgacga cctgctgtcc cggttcatgc 2160 ggaagggctc ctactcggac gagtcgctgc agcacgtggc gctcaacttc atcctcgcgg 2220 gccgcgacac atcctccgtg gcgctctcct ggttcttctg gctcgtgtcc acccaccccg 2280 ccgtggagcg caaggtcgtg cacgagctct gcgccgtgct cgcggcgtcc cggggcgccc 2340 atgacccggc attgtggctg gcggcgccct tcaccttcga ggagctcgac agtctggtct 2400 acctcaaggc g 2411 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Os02g44654-proF_kpn1 <400> 2 ggtacctagt agccgtatca cattac 26 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Os02g44654-proR_XhaI <400> 3 tctagacccg ccttgaggta gaccagac 28 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGUS1 <400> 4 aacgctgatc aattccacag 20

Claims (9)

A composition for inducing root-first expression of a foreign gene in a phosphate-deficient environment, comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a vector comprising an operably linked foreign gene. For inducing root-first expression of a foreign gene in a phosphate-deficient environment, comprising a transformed cell transformed with a vector comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a foreign gene operably linked thereto Composition. 3. The composition according to claim 1 or 2, wherein the induction of root-preferential expression in the phosphate-deficient environment is preferential expression at the distal end of the root, wherein the root-preferential expression is induced in the phosphate-deficient environment. The composition according to claim 1 or 2, wherein the composition for inducing root-first expression of a foreign gene in the phosphate-deficient environment is applied to rice ( Oryza sativa ), wherein the composition is for inducing root-first expression of a foreign gene in a phosphate-deficient environment. A transgenic terminal plant transformed with a composition for inducing root-first expression of a foreign gene according to any one of claims 1 or 2, wherein the foreign gene is preferentially expressed in the root of the terminal plant in a phosphate-deficient environment. The method of claim 5, wherein the monocot plant is rice (Oryza sativa ). Comprising: transforming a transgenic plant with a vector comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a foreign gene operatively linked thereto, in a phosphate-deficient environment, Wherein the transgenic plant is transformed into a transgenic plant. A method for the production of transgenic monocotyledonous plants, wherein the foreign gene is preferentially expressed in the root in a phosphate-deficient environment comprising the steps of:
(a) preparing a composition for inducing root-first expression of a foreign gene in a phosphate-deficient environment, comprising a vector comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a foreign gene operatively linked thereto step;
(b) introducing the composition into a terminal plant; And
(c) selecting the transgenic monocotyledonous plants to which the composition is introduced such that the foreign gene is expressed primarily in a phosphate-deficient environment. 9. The method according to claim 8, wherein the terminal lobe is rice (Oryza sativa).

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