KR101613366B1 - A promoter for ubiquitous gene expression in monocotyledones and use thereof - Google Patents

Abstract

The present invention relates to a promoter expressing in whole body of rice and its use.
The promoter of the present invention is a promoter that expresses in the whole body of rice, and is useful when it is intended to introduce a gene that determines important traits such as promoters such as ubiquitin1, actin1, and 35S, which are currently widely used, into rice, .

Description

[0001] The present invention relates to a promoter for inducing systemic expression of monocotyledonous plants and a use thereof.

The present invention relates to a promoter for inducing systemic expression of monocotyledons and uses thereof.

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 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 1980s, transgenic plants were obtained by using polyethylene glycol (PEG) and electroporation in protoplasts. In the late 1990s, gene gun utilization became popular, and in recent years, The use of Agrobacterium, which has been widely used in plants, is also widely used. Other methods include pollen pathways and microinjection methods.

The promoter can achieve the transformational purpose by locating the expression of the foreign gene only at the whole body of the plant or a specific tissue, and can be classified as follows according to its 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, (Cf. Reg. 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.

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 rice major storage protein gene has been widely used to induce seed-specific expression of monocotyledonous plants so far. Promoters that are mainly used to induce seed-specific expression include soybean-derived lectin promoter, cabbage-derived napin promoter and γ-tocopherol methyl transferase (γ-TMT) in Arabidopsis seeds. A patent application (Patent Application No. 10-2006-0000783) for seed-specific expression induction of carrot-derived DC-3 promoter and perilla derived oleosin promoter used in the study promoting the production of vitamin E by inducing gene expression . 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, it is a root specific expression promoter. Although there are no commercial examples yet, root-specific expression of pyruvate peroxidase (prxEa) has been isolated, and recently, it has been confirmed that the expression of maz genes (ibMADS) derived from sweet potatoes and ADP-glucose pyrophosphatase , AGPase) gene was isolated to induce a specific expression of the promoter in the root, leading to root-specific transient expression in carrot and radish, and was registered as a patent (Korean Registration No. 10-0604186, No. 10-0604191) There is a bar.

Fourth, other tissue-specific promoters such as leaves can 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.

In addition, although the development is currently underway, new promoters capable of controlling the location of gene expression more precisely according to the intention of the developer, for example, genes that should be expressed only in specific organs (petals, roots, leaves, stems, etc.) , Inactivated fertilization, burning, specific metabolism-related substances, defensive substances, etc.), there is a need to continue to develop promoters that operate only at specific institutions or periods.

Korean Patent No. 10-1080291 Korean Patent No. 10-0803391

The present invention relates to a transgenic plant, which comprises a promoter capable of expressing a foreign gene in the whole body of a terminal plant, an expression vector comprising the promoter, a transformed cell transformed with the expression vector, a progeny of a foreign gene A composition for inducing expression thereof, a terminal plant transformed with the same, and a method for producing the same.

In order to achieve the above object, the present inventors have sought to establish a system capable of introducing genes for determining important traits into rice or other plants by identifying promoters capable of inducing gene expression in the whole body of plants. In this process, it was confirmed that the promoter of LOC_Os04g42090 of rice was generally expressed in leaves, roots, ovaries, stomachs, pistils, seeds, etc. Thus, the promoter was secured and an expression vector containing the promoter was prepared, And confirming that the reporter gene is expressed in almost all the tissues of the transformed transformant, thereby completing the present invention.

More specifically, we constructed a database for meta-expression analysis by 17 organizations based on 983 rice microarray data published in the public database. Through analysis of this data, 4000 These genes were isolated. In order to search for and identify the promoters of these genes, the rice T-DNA gene trap vector system (pGA2707, pGA2715, pGA2717 pGA2772) was searched. As a result, strain 3A-05916 was inserted into the first intron of LOC_Os04g42090 gene, . The microarray meta gene expression analysis and GUS activity analysis in various rice organs and tissues confirmed that the promoter of this gene induces gene expression in almost all tissues of rice.

The gene expression pattern regulated by these genes was precisely analyzed by expressing the GUS reporter gene in the 3 'direction of these promoters and measuring its activity in the rice. Because of the time-consuming difficulty of transgenic vectors for the verification of promoter activity, the present inventors have utilized a fusion omics analysis technique, which has been recently actively pursued by a method different from the conventional method.

Since the GUS gene without the promoter is adjacent to the right end of the T-DNA of the promoter search vectors pGA2707, pGA2715, pGA2717 and pGA2772, when the T-DNA is inserted in the direction of the promoter 3 'in the forward direction, the promoter activity Can be measured. In addition, since the promoter trap system can confirm large-scale T-DNA insertion sites through genomic DNA PCR and base sequence analysis, it is possible to search the related system by indexing the gene locus id in the database.

The isolated 4000 whole body expression genes were selected as 224 promoter trap lines for 220 genes through the related promoter search system. The seeds of these lines were sterilized and the entire rice nipple was used for GUS analysis in the nutrient medium for 7 days.

As a result, in experimental group 3A-05916, T-DNA was inserted into the first intron of LOC_Os04g42090 gene which is not homologous with the existing gene, and GUS expression analysis and genomic DNA PCR showed that GUS expression in this lineage promoter activity . The promoter up to about 2kb in front of ATG of LOC_Os04g42090 was isolated by genomic DNA PCR and cloned into pGEM T-Ease vector and base sequence was decoded by referring to base sequence decode information. Based on this, a vector containing the LOC_Os04g42090 gene promoter and a transformant were prepared.

The present invention provides a promoter expressed in plant system comprising a promoter of LOC_Os04g42090 of rice, which is the nucleotide sequence of SEQ ID NO: 1.

A promoter is an essential element to regulate the environmental, temporal, and systemic expression of a gene. Using the systemic expression-inducing promoter according to the present invention, the expression of an exogenous gene of interest in plants can be induced in whole plants. The present invention provides a promoter of a rice LOC_Os04g42090 gene, which promotes the expression of a foreign gene in the whole body of a plant (hereinafter referred to as 'promoter of the present invention').

LOC_Os04g42090 encodes a conserved peptide uORF-containing transcript (CPuORF7) and consists of one exon. The promoter of this gene consisting of 398 amino acids has the nucleotide sequence of SEQ ID NO: 1.

The present invention also provides a plant systemic expression vector comprising the promoter and an operably linked foreign gene.

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. "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 resulting in the expression of the cloned DNA upon introduction into an appropriate host cell. 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 vector as long as it can introduce the promoter of the present invention. Preferably, the vector may be a vector such as pCAMBIA family, pGA family, pGWB family such as pGA3383, pCAMBIA3301, pCAMBIA3300, pGA3426, pGA3780, pGWB12, pGWB14 Ti-plasmid, and vectors derived therefrom. These vectors contain the promoter and foreign gene of the present invention and have a remarkable effect on foreign gene expression in plant system.

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.

Means a fragment or a fragment of a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1, which exhibits substantially equivalent effect to the promoter of the present invention. 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. In addition, the promoter represented by SEQ ID NO: 1 of the present invention is a sequence located in front of ATG of LOC_Os04g42090 on the published rice genome sequence, and even if some of the nucleotide sequences shown in SEQ ID NO: 1 are changed, added or deleted, It can be included in the scope of the present invention if homology with the sequence located in the promoter of LOC_Os04g42090 in the genome sequence is maintained and exhibits substantially equivalent effect.

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 intended to be expressed in plant whole systems. The foreign gene may be anything related to various characteristics for the purpose of expression of whole body of plants such as increase of productivity, increase of stress tolerance, and the like. For example, Low silicon rice 1 (Lsi1) in connection with productivity and growth of rice , and the present promoter can be used to systemically express a gene of interest such as a gene responsive to abiotic stress such as OsNAC10 .

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 a host include, but are not limited to, 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. "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 systemic expression of a terminal plant of a foreign gene comprising the expression vector or the transformed cell. The composition for inducing systemic expression of monocotyledonous plants may induce the expression of foreign genes in monocotyledonous whole plants by introducing a foreign gene into monocotyledonous plants. 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 systemic expression refers to expression in whole plant parts including leaves, stems, roots, nests, stomachs, pistils, seeds and the like of the terminal leaves.

The present invention also provides a transgenic plant transformed with the vector, the transgenic cell, or a foreign gene comprising the transgenic cell, wherein the transgenic plant is rice. These transgenic monocotyledonous plants exhibit the characteristic that the introduced foreign gene is expressed in the whole plant of the monocotyledonous plant.

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 whole plant of the terminal plant.

Transformation of the present invention terminal plant may be carried out by any method known in the art for transferring DNA to the terminal 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 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 0301316) and the like.

Such mutants can be easily produced by molecular biological methods well known in the art.

The present invention also encompasses a step of transfecting a monocotyledonous plant with a vector comprising a promoter comprising the nucleotide sequence of SEQ ID NO: 1 and a foreign gene operably linked thereto, and expressing the foreign gene in the monocotyledonous plant Which expresses a foreign gene in the whole body.

Preferably, the method comprises the steps of: preparing a composition for inducing systemic expression of a terminal plant of a foreign gene comprising the expression vector or the transformed cell; Introducing said composition into a terminal plant; And selecting the transgenic monocotyledonous plants to which the composition is introduced and the foreign gene is expressed in whole body of the monocotyledonous plant.

The promoter of the present invention is a promoter that is expressed in whole body of a terminal plant, and when a gene that determines important traits such as promoters such as ubiquitin1, actin1, and 35S which is currently widely used is to be introduced into a crop or a terminal plant including rice Can be usefully used.

FIG. 1 is a heat map showing the expression of the isolated gene showing expression in the whole body of rice.
FIG. 2 is a diagram showing a T-DNA insertion site for confirming the promoter activity of the present invention. FIG.
FIG. 3 is a diagram showing GUS staining results and PCR results showing that the 3A-05916 strain co-segregates with the LOC_Os04g42090 gene.
FIG. 4 is a diagram showing the results of confirming the systemic expression pattern of the LOC_Os04g42090 promoter in various tissues of the 3A-05916 strain through GUS staining.
FIG. 5 is a diagram showing microarray results showing systemic expression of the LOC_Os04g42090 gene. FIG.
6 is a diagram showing a diagram of a vector used for the production of a GUS plant containing the LOC_Os04g42090 promoter.
7 is a diagram showing an about 2 Kb promoter in front of ATG of LOC_Os04g42090 gene.
Fig. 8 is a graph showing the overall expression of GUS in various organs of transgenic rice plants in which a promoter of LOC_Os04g42090 was introduced.

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> Mass selection of rice genes expressed in whole body of rice

In order to screen genes expressing in plant whole systems, 983 kinds of rice epimetric microarrays (affymetrix (R)) collected from NCBI GEO (http://www.ncbi.nlm.nih.gov/geo/), an open microarray database microarray data were reconstructed by 17 organizations / organizations to construct a database for gene expression analysis.

This is shown in Table 1.

Table 1. Database for gene expression analysis reconstructed by institution / organization

Using the K-means clustering analysis method, a gene group having a high expression level in plant system among the database for gene expression analysis shown in Table 1 was isolated.

This was performed using the K-means clustering analysis method of MeV, a microarray data analysis software, and 4,034 genes with high expression in the plant system were isolated from the databases for expression / organization-specific expression analysis.

The results are shown in Fig.

As shown in FIG. 1, the closer to yellow, the higher the preferential expression is, and the closer to blue the gene has the lower priority. That is, among the 17 reconstructed gene databases, 4,034 genes having high expression in plant system could be identified.

< Example  2> Promoter for systemic expression gene trap  Identification of the system

Since the GUS gene without the promoter is adjacent to the right end of the T-DNA of pGA2707, pGA2707, pGA2715, pGA2717, and pGA2772, which are vectors for the promoter search, GUS expression is observed when the T- DNA is inserted in the forward direction and the forward direction of the promoter The promoter activity can be known.

Through inverse PCR and sequence analysis of T-DNA-inserted rice lines, T-DNA insertion sites were determined on the rice chromosome on a large scale. The mutation information of the T-DNA index corresponding to 1/2 of the genome was analyzed.

As a result, it was possible to search for a gene having a locus id name and potentially trapping the promoter from the above 4000 systematic expression genes. Through this, 224 promoter trap lines for 220 genes were screened.

< Example  3> GUS  Expression analysis of whole body expression gene by staining method

In order to confirm the organ specificity of the selected gene, 10 seeds of 224 promoter trap lines for 220 genes showing systemic expression were cultured at 28 ° C for 7 days, and all individuals were stained with GUS.

The GUS solution was prepared by mixing 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, 1 g of X-glucin dissolved in 10 ml of DMSO, 200 ml of distilled water was added to make 1 L volume. The rice of each line was treated with 15 ml of GUS staining solution for 24 hours and decolorized at room temperature using 70% ethanol and 100% ethanol sequentially.

As a result, GUS expression was observed in 20 lines among 200 lines subjected to GUS staining. Among them, thirteen strains with T-DNA insertion in the forward direction were selected.

< Example  4> Systems showing systemic expression GUS  Expression and T- DNA  Cover co -segregation verification

The selected 13 lines of rice seeds were cultured in MSO medium at 28 占 폚 for 7 days. 100 mg of leaves of the 7 day cultivated leaves were sampled and pulverized in liquid nitrogen, and a part of the leaves were used for GUS staining. DNA was extracted from the pulverized leaves using CTAB buffer (CTAB 10 g, NaCl 40.91 g, 0.5 M EDTA (pH 8.0) 20 ml, PVP 10 g and ascorbic acid 0.44 g, and 500 ml of water).

The selected 13 lines of rice seeds were cultured in MSO medium at 28 占 폚 for 7 days. Leaves of the cultivated individuals were collected, quenched into liquid nitrogen, ground, and partially used for GUS staining. DNA was extracted from the pulverized leaves using CTAB buffer (CTAB 10 g, NaCl 40.91 g, 0.5 M EDTA (pH 8.0) 20 ml, PVP 10 g and ascorbic acid 0.44 g, and 500 ml of water).

DNA was extracted from thirteen lines of rice leaves. Among them, 3A-05916 of the experiment group showed insertion of GUS reporter gene in the first intron of LOC_Os04g42090 gene and insertion position in FIG. 2. Genomic analysis was performed by PCR using primers (SEQ ID NOS: 2 and 3) and NGUS1 primers (SEQ ID NO: 4) prepared before and after the T-DNA insertion site, and the primers used were shown in Table 2 below.

Table 2. Primers 3A-05916

1 in FIG. 2 is nucleotides 1,161 to 2807 of SEQ ID NO: 5, and 1 in FIG. 2 is a sequence corresponding to exon.

After 5 minutes at 95 ° C, the PCR reaction was repeated 37 times at 95 ° C for 30 seconds, at 57 ° C for 30 seconds, at 72 ° C for 1 minute 30 seconds, and finally at 72 ° C for 5 minutes. The PCR reaction product was stored at 4 ° C and a part thereof was confirmed by electrophoresis.

The results are shown in Fig.

As a result, as shown in Fig. 3, it was confirmed that the PCR result of the experimental group 3A-05916 and the GUS staining result coincided with each other.

FIG. 3A shows results of GUS staining for 8 one-week-old individuals of 3A-05916, and FIG. 3B shows the genotype analysis results of the individuals used in experiment A of FIG.

The upper band in FIG. 3B is the PCR product of the gene specific primers (SEQ ID NOS: 2 and 3) of LOC_Os04g42090 and the lower band is the PCR product of SEQ ID NO: 3 and NGUS1 (SEQ ID NO: 4) on the RB PCR products. By comparing the results of the two products, genetic analysis of the plant is possible.

The amplified products of SEQ ID NOS: 2 and 3 are wild type and the products of SEQ ID NOS: 2 and 3 and the products of SEQ ID NOS: 3 and 4 are hybrid, When the product is amplified, it means homo.

In the wild type, GUS expression is not observed because there is no T-DNA insertion. GUS expression on T-DNA is observed in hybrids and purebreds.

That is, it was confirmed that the 3A-05916 strain was co-segregated with the LOC_Os04g42090 gene.

In addition, as shown in Fig. 4, GUS expression was observed in the whole body even in the result of 7-day staining of 3A-05916 strain. As shown in Fig. 4, it was confirmed that the transgenic plants showed GUS expression in the whole tissue. Each of Fig. 4 represents the following tissues of a plant: (a) a young leaf, (b) a mature sheet, (c) a section of a mature sheet, (d) a spikelet before flowering, (e) (G) Young roots (h) Seminal root section of young roots (i) Cross section of crown root of young roots, showing GUS expression in all tissues there was.

< Example  5> Expression analysis of genes by microarray data analysis

The expression pattern of LOC_Os04g42090 gene was analyzed by microarray database analysis and the results are shown in FIG.

As shown in Fig. 5, it can be confirmed that all tissues are yellowish, indicating that the expression of the gene by the LOC_Os04g42090 promoter occurs in the whole plant system.

Based on the results of FIGS. 3 to 5, 3A-05916 showed that the experimental group 3A-05916, in which the GUS reporter gene was linked to the first intron of the LOC_Os04g42090 gene, was expressed in plant system.

< Example  6> Promoter for systemic induction expression Cloning

PCR was performed using the primers prepared to clone the promoter of LOC_Os04g42090 and 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 55 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 BamH1 and Xho1, followed by ligation with pGA3383 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 50 占 퐇 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 BamHI and XhoI, 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). Primers using NGUS1, RB (right border), and promoter sequences were used for the nucleotide sequence analysis. A diagram of the vector used for the production of the LOC_Os04g42090 promoter GUS plant thus constructed is shown in Fig.

Primer for promoter cloning is shown in Table 3 below.

Table 3. Primers for promoter cloning of LOC_Os04g42090

Table 4. Primers for sequencing

This sequence is shown in FIG. 7 and SEQ ID NO: 1, and corresponds to a promoter sequence corresponding to about 2000 bp in front of the ATG of the LOC_Os04g42090 gene.

< Example  7> Production of transformed cells

The rice expression vector prepared in Example 6 was extracted.

50 아 of Agrobacterium tumefaciens LB4404 and 2 의 of rice expression vector 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. Next, the cells were plated on tetracycline-resistant LB solid medium and incubated for 36 hours. The resulting colonies were cultured in 1 mL of LB liquid medium, mini-preparations were performed, and BamH1 and Xho1 were used for enzyme cleavage.

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

< Example  8> Production of transgenic rice

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 rice expression vector obtained in Example 6 and left in a 22 ° C cancer-treated growth chamber in a medium containing N6D-Acetosyringone.

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. After induction of regeneration in the light condition growth chamber for 4 weeks at 28 ° C., the rice was transferred to MS solid medium, grown in a light condition growth room for 7 days, And raised the regrowth rice.

< Example  9> Verification of plant system expression using transgenic plants

In order to confirm the organ specific GUS expression of regenerated plants, samples were taken at various times and tissues and GUS staining was performed to confirm GUS expression in plant system.

The results are shown in Fig. 8 (a), 8 (b), and 10 (c) show cross-sections of mature seeds, mature leaves and (e) spikelet before flowering. . As shown in FIG. 8, it was confirmed that the expression of GUS in the transformant and the promoter trap system 3A-05916 in which the LOC_Os04g42090 gene promoter was introduced showed in the whole tissue of rice.

< Example  10> LOC _ Os04g42090   Gene RNA - seq  Expression analysis

The results of analysis of RNA-seq expressing systemic expression of the LOC_Os04g42090 gene are shown in Table 5.

Table 5. RNA-seq expression analysis of LOC_Os04g42090 expression in whole plants

The above data is based on the gene expression analysis service (http://rice.plantbiology.msu.edu/expression.shtml) provided by the Rice Genome Annotation Project team of Michigan State University. The data used in the expression analysis is the next generation sequence analysis Technology-based RNA-seq analysis. The frequency of RNA-seq identified for each library listed in Table 5 indicates the degree of expression of the examined gene. This gene, as a result of RNA-seq analysis, showed a high level of expression in the whole tissue. Compared with other organs, expression was higher in whole tissues. This confirms the expression patterns observed in the microarray.

<110> University-Industry Cooperation Group of Kyung Hee University <120> A promoter for ubiquitous gene expression in monocotyledones and          use thereof <130> P13-078-REA-KHU <150> KR 2013/0122555 <151> 2013-10-15 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 1907 <212> DNA <213> Oryza sativa <220> <221> promoter <222> (1) (1907) <223> LOC Os04g42090 promoter <400> 1 cgcccacaca aattcgtgtg gttttgggca aacgcagccg tgagcttttt tttttttttt 60 ccctgaagct cctgtgtgtc atctctcttt ttcttttttg agctagaatt ggcaaaactt 120 ttttaatccg gtgtcttttc atccgtgttc ctgtgaaagc agcgccgaaa actacccccg 180 ctttgcaaaa agaaaaaaat cctctcgcgc tttgtttttt tttttttttt ttttgccttt 240 gcgacctcat cggcatccgc tttgcgaccc ggccgatgaa gagggggacc ggggagacgg 300 acggacgctg tgagccgcga atcccaccag cgcgtgtggg cccacctgcc acgggtgacg 360 cgccggccgg tccagcctcc acgcgagcag cgggtcgggc gcgtcccctc tccttggcgg 420 tcacgaacac tgaaaaaccc gaatccgata agcggccgct ataaataccg ggccaaatca 480 gggtttccct tccccagccg caccgcacgc tttcttgcat aagaactccc ccccctcctc 540 gctccccgcc gccgccgccg ttttcttgga gaggagaaat ccgtaggctc agaggatttt 600 tggtagtctt gtttcgtgat attcgtgaag agggggggag ctaagggggg attcgtaaga 660 tcagaattgg atcacgcggg ggcgcgctcg ggatcggggg ttccaaacac agcttcttcg 720 ttgaattgta agtttcttcg ctgaaaccct ctccgtaggt tggtacttct gatttttttt 780 tcctttacag attttgtggt ttgtagggcg tattgctatg tgttgctatt gtttatgatg 840 ggaattttgg tttgttggtg tagataagct tatctatgta aaacaaaagg tcagtaggtt 900 acctctttga tcttgcttgt gcagttaggc tatcctgttt gagttgttcg tgatttgcag 960 attactgcaa aagctatcct gtaaaatttc ttgtcgtggt aaagattgta ctaagaattt 1020 ttgtgtgttc agatcctaag acatagaaaa atagtttttt ttcgtctccc cataggaata 1080 taacaaatgt tctttttctg tgacaccatg atcgaatttt gttttttaat gcaaatgtat 1140 ggtattatca ttaacatagg atgggatcag ggacgaaaca tatgtgaata taggaacatt 1200 tgcatatgtc caacggaaaa gtctcaactc taaatcttaa tccaaatgac atcacgattg 1260 taaatccgac cagatcttaa atctgaatcc aagtgagact caattgtaaa tctaaatata 1320 agaaagaata ctgatatatg attcattgta aaggacgctg tggcttgtag gaattgagta 1380 ttactggaaa attaggaatt gaatcagatt gtgagatatt aattgacctg tcctgttgtg 1440 gcatatttac tcctagttag aacttggaat ctatcatatt gttatgtaac tgtttactct 1500 ttggtatcgt tttgatagct tcttatctca gttttacttt tttgcattaa agactcagat 1560 gcttcggtct ggcacatata tcttctacaa acatatgccc ttattttctc tttgggccat 1620 atatatttgt ctgaacctga tatttacctt gttatatact tgtgcagtaa gtggatgtac 1680 taatggagtc aaagggtggc aaaaagaagt ctagcagtag tcgttccttg atgtatgaag 1740 ctccccttgg ctacagcatt gaggacgttc gacctgccgg aggcgtgaag aagttccagt 1800 ctgctgctta ctccaacgta agcatcaaat acagatttaa ttccctcctt tgtatcttcc 1860 gatgtgttgc taatcatgtc aattgttcct tttgttgcag tgcgcga 1907 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3A-05916-Forward primer <400> 2 ctttctcctt gtccagtccg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 3A-05916-Reverse primer <400> 3 taatggagtc aaagggtggc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NGUS1 <400> 4 aacgctgatc aattccacag 20 <210> 5 <211> 3713 <212> DNA <213> Oryza sativa <400> 5 tccccagccg caccgcacgc tttcttgcat aagaactccc ccccctcctc gctccccgcc 60 gccgccgccg ttttcttgga gaggagaaat ccgtaggctc agaggatttt tggtagtctt 120 gtttcgtgat attcgtgaag agggggggag ctaagggggg attcgtaaga tcagaattgg 180 atcacgcggg ggcgcgctcg ggatcggggg ttccaaacac agcttcttcg ttgaattgta 240 agtttcttcg ctgaaaccct ctccgtaggt tggtacttct gatttttttt tcctttacag 300 attttgtggt ttgtagggcg tattgctatg tgttgctatt gtttatgatg ggaattttgg 360 tttgttggtg tagataagct tatctatgta aaacaaaagg tcagtaggtt acctctttga 420 tcttgcttgt gcagttaggc tatcctgttt gagttgttcg tgatttgcag attactgcaa 480 aagctatcct gtaaaatttc ttgtcgtggt aaagattgta ctaagaattt ttgtgtgttc 540 agatcctaag acatagaaaa atagtttttt ttcgtctccc cataggaata taacaaatgt 600 tctttttctg tgacaccatg atcgaatttt gttttttaat gcaaatgtat ggtattatca 660 ttaacatagg atgggatcag ggacgaaaca tatgtgaata taggaacatt tgcatatgtc 720 caacggaaaa gtctcaactc taaatcttaa tccaaatgac atcacgattg taaatccgac 780 cagatcttaa atctgaatcc aagtgagact caattgtaaa tctaaatata agaaagaata 840 ctgatatatg attcattgta aaggacgctg tggcttgtag gaattgagta ttactggaaa 900 attaggaatt gaatcagatt gtgagatatt aattgacctg tcctgttgtg gcatatttac 960 tcctagttag aacttggaat ctatcatatt gttatgtaac tgtttactct ttggtatcgt 1020 tttgatagct tcttatctca gttttacttt tttgcattaa agactcagat gcttcggtct 1080 ggcacatata tcttctacaa acatatgccc ttattttctc tttgggccat atatatttgt 1140 ctgaacctga tatttacctt gttatatact tgtgcagtaa gtggatgtac taatggagtc 1200 aaagggtggc aaaaagaagt ctagcagtag tcgttccttg atgtatgaag ctccccttgg 1260 ctacagcatt gaggacgttc gacctgccgg aggcgtgaag aagttccagt ctgctgctta 1320 ctccaacgta agcatcaaat acagatttaa ttccctcctt tgtatcttcc gatgtgttgc 1380 taatcatgtc aattgttcct tttgttgcag tgcgcgaaga agccatcctg atagcccttt 1440 cggcttctcc atcctagtag tttaggattt cttgcaattc cattttggca cttttcttct 1500 gacctatttc tctggctgct gcttcctgat aatcgaccag ttctctagtc ttgctccctg 1560 cactcctccc tcctccatct ccggcacagt gttctgacca acctgctcca atgggtgtct 1620 tgtctgctgc tgaccctccc ccagtctcag caattgggtt tgagggctat gagaagcgcc 1680 ttgagatcac tttctctgag gcacctgtct ttgctgaccc tgatggtcgg ggtttgcgcg 1740 ccctctccag ggcccagatt gactctgttc tggatcttgc acggtgcacc attgtgtccg 1800 agctgtccaa caaggacttt gactcctatg tcctctctga gtccagcctg tttatctatt 1860 ctgataagat tgtgattaag acctgtggga ctacaaagct cctgctcaca attccaagga 1920 ttcttgagct tgctgaaggg ctttctatgc cacttgctgc tgtgaagtac tcccgcggga 1980 tgttcatctt ccccagtgca cagcctgctc cccacaggag cttctctgag gaagttgctg 2040 tcctcaaccg ctactttggc catctgaaat ctggtggtaa tgcttatgtg attggagatc 2100 cagcaaagcc tggccagaag tggcatatct actatgctac tcagcacccg gagcaaccta 2160 tggttaccct tgaaatgtgc atgaccggac tggacaagga gaaagcttca gtctttttca 2220 agacttctgc tgatggtcac acatcatgtg ctaaggaaat gacaaaactc tcgggcatct 2280 ctgacattat cccagagatg gagatctgtg actttgactt cgaaccctgc ggctactcca 2340 tgaatgcaat ccatggctca gcgttttcta ccattcatgt gacccctgag gatggcttca 2400 gt; tgaagagggt cctcaggtgc tttggccctt cggagttctc tgttgctgtt accatctttg 2520 gtgggcatgg tcatgctgga acatgggcaa aggagctcaa tgctgatgct tacaaatgca 2580 acaacatggt agagcaggag ctgccctgtg gtggcctcct catctaccag agcttcgacg 2640 ctactgaaga cgtacctgtt gctgttgggt cgcccaaatc tgttctgcac tgcttcgagg 2700 ctgagaatat ggtgaaccct gctcctgtta aggaaggtaa actgggcaat cttctcccct 2760 ggggagagga tgcgttggag gagaatgatg gagtgtttga tgagtaagat gggacttctg 2820 gtgacgattc gctgctttgt tttcattccg tggcattttt atctgttgag gttgtcgttt 2880 ggttattctg tgaagcagcc agccaggcta ttagtatttt cgcttgtaag catgtgttct 2940 gatcttggga cgtatgcttg gtctaaataa taatggtgaa ctaaaagctc taggtggtca 3000 gctgcgtctg ccacaatgag catgcgtcgg gagaaatgtt gtcaaaacat atgaataagt 3060 atttgcggga aatttccttt gccttctgct aatgttcctt ccgatctcaa ttgttaaatt 3120 ttgttgctgc tggtgtctaa gaaagttctg cttgggacac tagatagtat ggagaaaacg 3180 attttcgaaa gtttccactc ttgttatctc aactctgtag agctccccct gttccatggg 3240 actgcaaatg cccatggcac aggtatatct taaggtagta cattaatgaa aagtctggga 3300 gaatagcatg tgcaaaatgc ttactagctc ttataagtta aaaatagctg gaagaactgc 3360 attctatagc tttccccgtg ttcatggtta tcttctgcaa gatatggctg gacgaagtat 3420 atgccgcatg cttggctctt gccgccccat tcgccttggc atggaccatt gacttcgatt 3480 ttttttttgg acatgtttat gtatttgctt attgtcctct actttacatg taacagtaat 3540 ttaaaggagt gctgcctgat ctgataattg cgtttggaat cctttaatga gcttgatctt 3600 gtcgcgcttg gaaagatgat gcagcgacct gttcggtacc ctatgcttat cttctgacat 3660 ccatatctgt attattgttt atgaagttgt acgctttgta cccaattatc cat 3713 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Os04g42090-BamHI-Forward primer <400> 6 ggatcccgcc cacacaaatt cgtgtg 26 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Os04g42090-Xho1I-Reverse primer <400> 7 ctcgagtcgc gcactgcaac aaaagg 26 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > RB_backbone-F2 <400> 8 tcgcacggaa tgccaagca 19 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Os04g42090-seq_1 <400> 9 attcgtaaga tcagaattgg 20

Claims (8)

1. A composition for inducing systemic expression of a transgenic plant of a terminal plant, comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a vector comprising a foreign gene operatively linked thereto. 1. A composition for inducing systemic expression of a transgenic plant of a terminal plant comprising a transgenic cell transformed with a vector comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and an operatively linked foreign gene. The composition according to claim 1 or 2, wherein the terminal plant is a rice plant (Oryza sativa). A transgenic terminal plant transformed with a composition for inducing systemic expression of a terminal plant of the foreign gene of claim 1 or 2 and expressing the foreign gene in the whole plant of the terminal plant. The transgenic plant of claim 4, wherein the terminal plant is rice (Oryza sativa). Comprising the step of transfecting a monocotyledonous 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, and expressing the foreign gene in the monocotyledonous plant, A method for producing a transgenic terminal plant expressing in whole body. A method for producing a transgenic monocotyledonous plant, wherein a foreign gene is expressed in the whole body of the monocotyledonous plant comprising the steps of:
Preparing a composition for inducing systemic expression of a terminal plant of a parent plant, comprising a promoter consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a vector comprising a foreign gene operatively linked thereto;
Introducing said composition into a terminal plant; And selecting the transgenic monocotyledonous plants to which the composition is introduced and the foreign gene is expressed in whole body of the monocotyledonous plant. 8. The method according to claim 7, wherein the terminal plant is rice (Oryza sativa).

Images