# Patent

## Title
Promotes and methods thereof

## Abstract

A promoter, which may be used to transform a plant and/or express a gene substantially uniformly in substantially all organs and/or tissues of a plant, and which may include a constitutive expression promoter for transforming a monocot plant. A vector including a promoter, which may include a recombinant plant expression vector. A method of producing a target protein using a vector, and a method of producing a transformed cell and/or plant using a vector. A transformed plant, a transformed seed and a transformed cell are included, which may be formed by the method of producing the same using a vector. A PCR primer for a promoter is provided.

## Background
BACKGROUND
The present invention generally relates to promoters and methods of use and fabrication thereof, and particularly to a promoter used to express a gene, a vector including a promoter, a method of producing a target protein, a method of producing a transformed cell and/or a plant, a transformed plant, a transformed seed, a transformed cell, and PCR primers for a promoter.
A promoter may relate to a genomic region located upstream of a structural gene and may function in the transcription of a structural gene, for example, into mRNA. A promoter may be activated by binding of general transcription factors, and may include base sequences such as a TATA box and/or CAT box which may assist to regulate gene expression. For example, promoters linked to genes may be constitutively activated by general transcription factors to express genes associated with proteins needed for the basal metabolism of a living organism and which may be required in cells at a given concentration. Promoters may also be activated when proteins are needed which are not ordinarily present or only required under special circumstances. For example, inducible promoters may be activated by binding of specific transcription factors, which may be activated in an organism's developmental processes or by external stimuli resulting from surrounding environmental factors.
A foreign gene (i.e., transgene) introduced into a plant, forming a plant having novel characteristics which may develop an agricultural field, may be influenced by transcriptional, post-transcriptional, translational and post-translational elements. A promoter may belong to a transcriptional element and may directly influence transcription of a transgene, for example, to change the expression level of a transgene. A promoter may be the most important factor to change the expression stage or the tissue and/or cell specificity of a transgene.
Although promoters have been isolated from plants to express a transgene, only a few promoters may be practical for use in the transformation of plants. For example, a CaMV (cauliflower mosaic virus) 35S promoter and its derivatives may induce expression of genes in plant tissues and exhibit high activity, for example in vascular tissues and root/leave cells. However, a CaMV 35S promoter has relatively less activity in monocot plants, such as a rice plant, and does not exhibit any activity in certain cells, such as pollen.
Promoters from dicot plants which have been investigated for the transformation of monocot plants have exhibited relatively lower activity compared to promoters originating from monocot plants. A rbcS (ribulose bisphosphate carboxylase/oxygenase small subunit) promoter of rice, a Act1 (actin1) promoter of rice, and a Ubi1 promoter of maize are examples of promoters from monocot plants which have been investigated in the transformation of monocot plants. While Act1 and Ubi1 promoters exhibit a relatively high activity in monocot plants compared to a CaMV 35S promoter, there are drawbacks. For example, the Act1 promoter exhibits activity mainly in vegetative tissue and reproductive tissue, and thus is not effective for expression of a ubiquitous gene in monocot plants. Although the Ubi1 promoter exhibits activity in numerous types of cells, it does not exhibit activity in the substantially all tissues of a plant. Also, while the Ubi1 promoter exhibits a strong activity, especially in young roots, the activity is greatly reduced over time, for example as the root grows.
Accordingly, there is a need for developing a promoter exhibiting a strong, stable and ubiquitous activity in the transformation of plants, including monocot plants. There is a need for suitable promoters useful in the substantially uniform expression of a gene in substantially all the tissues of a plant. There is a need for suitable promoters useful in the production of transformed compositions, and suitable methods for fabricating the same. There is also a need for suitable primers for a variety of novel promoters.
SUMMARY
Embodiments relate to a promoter. In example embodiments, a promoter may include at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 (i.e., SEQ ID NO: 1 through SEQ ID NO: 20).
Embodiments relate to a promoter that may be a constitutive expression promoter and may be configured to transform a plant, such as a monocot plant. In example embodiments, a monocot plant may include at least one of a rice, barley, wheat, maize, millet and Indian millet. In embodiments, a promoter may express a gene substantially uniformly in substantially all organs and/or tissues of a plant.
Embodiments relate to a promoter that may include a base sequence complementary to at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In embodiments, a promoter may include a variant of at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In embodiments, a promoter may include a base sequence complementary to a variant of at least one of SEQ ID NO: 1 through SEQ ID NO: 20.
Embodiments relate to a vector. In example embodiments, a vector may include a promoter comprising at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In embodiments, a vector may be a recombinant plant expression vector and may include a constitutive expression promoter configured to transform a plant, such as a monocot plant. In embodiments, a target gene downstream of a promoter encoding a target protein may be operably linked. In embodiments, a promoter of a vector may expresses a gene substantially uniformly in substantially all organs and/or tissues of a plant. In embodiments, a vector may include a variant of at least one of SEQ ID NO: 1 through SEQ ID NO: 20.
Embodiments relate to a transformed plant. In example embodiments, a plant may include a promoter comprising at least one of SEQ ID NO: 1 through SEQ ID NO: 20. Embodiments relate to a transformed seed. In example embodiments, a seed may include a promoter comprising at least one of SEQ ID NO: 1 through SEQ ID NO: 20. Embodiments relate to a transformed cell. In example embodiments, a cell may include a promoter comprising at least one of SEQ ID NO: 1 through SEQ ID NO: 20.
Embodiments relate to a method of forming a target protein. In embodiments, a plant may be transformed using a vector that may include a promoter which may include at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In example embodiments, a target protein may include at least one of interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin.
Embodiments relate to a method of transforming a cell and/or plant. In embodiments, a plant cell may be transformed using a vector that may include a promoter which may include at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In example embodiments, a transformed plant may be redifferentiated from a transformed plant cell.
Embodiments relate to a PCR primer. In embodiments, a primer may include at least one of SEQ ID NO: 67 through SEQ ID NO: 106. In example embodiments, a primer may react with a promoter including at least one of SEQ ID NO: 1 through SEQ ID NO: 20. Embodiments relate to a primer including a base sequence complementary to at least one of SEQ ID NO: 67 through SEQ ID NO: 106. Embodiments relate to a primer including a variant of at least one of SEQ ID NO: 67 through SEQ ID NO: 106. Embodiments relate to a base sequence complementary to the variant at least one of SEQ ID NO: 67 through SEQ ID NO: 106.



DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 shows the expression of genes according to embodiments relative to related promoters (OsCc1, Act1 and Ub1) in various tissues of a plant.
FIG. 2 is a schematic diagram of a vector according to embodiments.
FIG. 3 shows structures of promoters according to embodiments.
FIG. 4 shows results of Green Fluorescent Protein expression levels using RT (reverse transcription)-PCR in seeds of a transformed plant according to embodiments as well as the leaf and root tissues of 5-day-old, 20-day-old and 30-day-old the plants.
FIG. 5 and FIG. 6 show results obtained using real time-qPCR and quantitatively comparing the expression of Green Fluorescent Protein in seeds and flowers of a transformed plant according to embodiments, as well as the leaf and root tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old of the plants.
FIG. 7 is a graphic diagram showing promoter activity according to embodiments and predicted by quantitatively comparing Green Fluorescent Protein expression in several events.
FIG. 8 shows the expression of Green Fluorescent Protein fluorescence in a leaf and root of a transformed plant according to embodiments.
FIG. 9 shows the expression of Green Fluorescent Protein fluorescence in a seed of a transformed plant according to embodiments.
FIG. 10 shows the expression of Green Fluorescent Protein fluorescence in a flower of a transformed plant according to embodiments.



DESCRIPTION
Embodiments relate to a promoter derived from monocot plants, such as rice. In embodiments, the promoter may be suitable for the transformation of plants including monocot plants and may be suitable for the constitutive expression of plant genes. A promoter according to embodiments may express a gene substantially uniformly in substantially all the organs and/or tissues of a plant. In embodiments, a promoter may include at least one of SEQ ID NO: 1 through SEQ ID NO: 20 and may include base sequences complementary to the base sequences of SEQ ID NO: 1 to SEQ ID NO: 20. “Complementary” may relate to hybridization and/or base pairing between nucleotides or nucleic acids, for instance, between two strands of a DNA molecule.
A promoter may relate to a DNA molecule to which RNA polymerase binds in order to initiate transcription and may refer to a DNA region upstream of a structural gene. A plant promoter may relate to a promoter which may initiate transcription in a plant cell. A constitutive promoter may relate to a promoter which may be active in most environmental conditions and/or development states and/or cell differentiation states. Since the selection of a transformant may be carried out by various tissues at various stages, a constitutive promoter may be preferable. However, one is not limited to selecting a constitutive promoter according to embodiments.
In embodiments, a promoter may include at least one of an ascorbate peroxidase (APX) promoter of SEQ ID NO: 1, an AP2 domain containing putative gene (SCP1) promoter of SEQ ID NO: 2, a cytosolic phosphogluconate dehydrogenase (PGD1) promoter of SEQ ID NO: 3, a putative R1G1 domain containing protein (R1G1) promoter of SEQ ID NO: 4, a translation initiation factor EIF-5A (EIF5) promoter of SEQ ID NO: 5, a PF1 promoter (60S acidic ribosomal protein P1) of SEQ ID NO: 6, a PF2 promoter (translationally controlled tumor protein) of SEQ ID NO: 7, a PF3 promoter (probable aquaporin of SEQ ID NO: 8, a PF4 promoter (40S ribosomal protein S8) of SEQ ID NO: 9, a PF5 promoter (ubiquitin-conjugating enzyme OsUBC5b) of SEQ ID NO: 10, a PF6 promoter (ribosomal protein S26E family protein of SEQ ID NO: 11, a PF7 promoter (60S ribosomal protein L18) of SEQ ID NO: 12, an SC1 promoter (peptidyl-prolyl cis-trans isomerase) of SEQ ID NO: 13, an SC2 promoter (histone H3.3) of SEQ ID NO: 14, an SC3 promoter (glyceraldehyde-3-phosphate dehydrogenase) of SEQ ID NO: 15, an SC4 promoter (heat shock protein 82) of SEQ ID NO: 16, an SC5 promoter (60S ribosomal protein L9) of SEQ ID NO: 17, an SC6 promoter (plectin S10) of SEQ ID NO: 18, an SC7 promoter (histone H2B.1) of SEQ ID NO: 19, and an SC8 promoter (histone H2B.1) of SEQ ID NO: 20.
In embodiments, a promoter may include at least one of SEQ ID NO: 1 through SEQ ID NO: 20 and may be configured to transform a plant, including a monocot plant. In embodiments, a monocot plant may be, but is not limited to, at least one of a rice, barley, wheat, maize, millet and Indian millet. In embodiments, a promoter may express a gene substantially uniformly in substantially all the organs and/or tissues of a plant, including a monocot plant.
Embodiments relate to variants of a promoter which may include a variant of at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In embodiments, a variant may have different base sequences but include functional characteristics similar to those of at least one of SEQ ID NO: 1 through SEQ ID NO: 20. A variant may result from at least one of a substitution, deletion and insertion of nucleic acid base(s), or combinations thereof, including functional fragments thereof. In example embodiments, a base sequence complementary to the variant at least one of SEQ ID NO: 1 through SEQ ID NO: 20 may be included.
In example embodiments, a variant of a promoter may have a sequence identity of at least 70%, preferably at least 80%, even more preferably 90%, and most preferably at least 95% to the at least one of SEQ ID NO: 1 through SEQ ID NO: 20. A percentage of sequence identity to a polynucleotide may be determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) and/or substitutions as compared to the reference sequence (which does not include additions, deletions or substitutions) for optimal alignment of the two sequences.
Substantial identity of polynucleotide sequences may relate to a polynucleotide including a sequence having between 50-100% sequence identity, preferably at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 90%, and most preferably at least 95%. Substantial identity may also relate to when two nucleotide molecules are hybridized specifically to each other under a stringent condition such that their sequences may be substantially identical to each other. For example, stringent condition may vary depending on nucleotide sequences, and thus can be different at a different condition. At certain ionic strength and pH, for example, a stringent condition may be selected to have a temperature that is about 10° C. lower than the heat-melting point (Tm) of a specific sequence. Tm may relate to a temperature at which 50% of a target sequence is hybridized to a fully complementary probe (under the condition of certain ionic strength and pH). For example, a stringent condition for carrying out Southern blot analysis may include washing with 0.2×SSC at 65° C. For an oligonucleotide probe, washing may be carried out with 6×SSC at 42° C.
Embodiments relate to a vector. A vector may relate to a DNA fragment(s) and/or nucleotide molecules delivered to a cell. A vector may replicate DNA and be independently reproduced in a host cell. The terms “delivery system” and “vector” may be interchangeably used. An expression vector may relate to a recombinant DNA molecule including a desired coding sequence and other appropriate nucleotide sequences that are essential for the expression of a coding sequence in a specific host organism, such an operatively-linked coding sequence. The aforementioned other appropriate sequences may include at least one of an promoter, an enhancer, a termination signal and a polyadenylation signal that may be suitable for use in a eukaryotic cell. For example, any related terminator may be used according to embodiments. Examples thereof include, but are not limited to, nopaline synthase (NOS), rice α-amylase RAmyl A terminator, phaseoline terminator, and a terminator for optopine gene of Agrobacterium tumefaciens. Since a terminator region may increase the reliability and efficiency of transcription in plant cells, a terminator may be highly preferable.
“Recombinant” may relate to a cell which replicates a heterogeneous nucleotide and/or expresses the nucleotide, a peptide, a heterogeneous peptide, or a protein encoded by a heterogeneous nucleotide. A recombinant cell may express a gene or a gene fragment in a sense or antisense form, which are not found in the natural state of the cell. Embodiments relate to a vector which may be a recombinant vector.
Embodiments relating to vectors which may be used to introduce DNA into a plant host may include viral vectors, for example, non-integrative plant viral vectors, such as derivable from the double stranded plant viruses (for example, CaMV) and single stranded viruses, gemini viruses and the like. The use of such vectors may be preferable, particularly when it is difficult to stably transform a plant host.
Embodiments relating to vectors may preferably include at least one selective marker. A selective marker may relate to a nucleotide sequence having a property which allows selection based on a common chemical method. It may be any kind of gene that may be used for the differentiation of transformed cells from non-transformed cell. Examples thereof include, but are not limited to, herbicide-resistant genes, such as glyphosate or phosphintricin, and antibiotic-resistant genes, such as kanamycin, G418, bleomycin, hygromycin or chloramphenicol.
In example embodiments, a recombinant plant expression vector may include a Ti-plasmid vector that, when present in a suitable host, such as Agrobacterium tumefaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell. Embodiments include different types of Ti-plasmid vectors, for example those disclosed in EP 0 116 718, to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes. Particularly preferred forms of Ti-plasmid vectors are the so-called binary vectors as described in EP No. 0 120 516 B1 and U.S. Pat. No. 4,940,838.
In embodiments, a vector may be a recombinant plant expression vector including a promoter in accordance with embodiments. An example of the recombinant plant expression vector may be, but is not limited to, a vector shown in FIG. 2. As shown in the example embodiment of FIG. 2, a modified green fluorescent protein (GFP), a protease inhibitor II terminator gene (TPINII), an OsCc1 promoter (Pcytc), a herbicide-resistant gene Bar (phosphinotricine acetyltransferase gene) and a nopaline synthase terminator (TNOS) may be operably linked to a promoter according to embodiments. Also as shown in the example embodiment of FIG. 2, a MAR sequence may be attached to the terminal of the right-border sequence. In embodiments, the aforementioned attachment may minimize the change in expression in the chromosome in various sites of the chromosome such that only the inherent activity of the promoter according to embodiments can be measured.
In embodiments a vector, such as recombinant plant expression vector, may be prepared by operably linking a target gene encoding a target protein downstream of a promoter according to embodiments. “Operably linked” may relate to the element of an expression cassette which functions as a unit to express, for example, a heterogeneous protein. In example embodiments, a promoter that may be operably linked to a heterogeneous DNA which may encode a protein may promote the production of functional mRNA corresponding to the heterogeneous DNA.
In embodiments, a target protein may be any kind of protein, and examples thereof include, but are not limited to, proteins having medical utility, such as enzymes, hormones, antibodies or cytokines, and proteins which can accumulate large amounts of nutrients capable of improving the health of animals including humans. Example embodiments of a target protein include, but are not limited to, interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin.
Embodiments relate to a method of producing a target protein. In embodiments, the target protein may be produced by transforming a plant using a vector including a promoter in accordance with embodiments. In example embodiments, the target protein may be produced by constitutive expression in a plant and may include transforming a plant with a recombinant plant expression vector. Embodiments of a target protein are described hereinabove.
According to embodiments, plant transformation may refer to any method of introducing DNA is into a plant. Such transformation methods do not necessarily have a period for regeneration and/or tissue culture. In embodiments, transformation of a plant species is possible for dicot plants and for monocot plants. In embodiments, any transformation method can be used to introduce a hybrid DNA according to embodiments to a suitable ancestor cells. Example methods include a calcium/polyethylene glycol method for protoplast transformation, electroporation of protoplasts, microinjection into plant material, (DNA or RNA-coated) particle bombardment of various plant materials, gene gun methods, infection with (non-integrative) viruses, in planta Agrobacterium tumefaciens-mediated gene transfer by infiltration of adult plants or transformation of mature pollen or microspores (EP 0 301 316) and the like. A preferred method according to embodiments may include Agrobacterium-mediated DNA transfer. More preferably, use of a so-called binary vector technology, disclosed in EP A 120 516 and U.S. Pat. No. 4,940,838, may be used.
According to embodiments, a plant cell that may be used in plant transformation may be any plant cell. A plant cell may be a cultured cell, a cultured tissue, a cultured organ or a whole plant, and may preferably be a cultured cell, a cultured tissue or a cultured organ, and most preferably a cultured cell according to embodiments. Plant tissue may relate to a differentiated or undifferentiated plant tissue. Embodiments of a plant tissue include, but are not limited to, root, stem, leaf, pollen, seed, cancerous tissue, and cells of various shapes that are used in culture, namely, single cells, protoplasts, buds and callus tissues. In embodiments, a plant tissue may be in planta or in organ culture, tissue culture or cell culture.
Embodiments relate to a method of producing a transformed cell and/or plant. In embodiments, the method may include transforming a plant cell with a vector including a promoter according to embodiments, and may include differentiating a transformed plant from a transformed plant cell. In example embodiments, a recombinant plant expression vector may be used. In example embodiments, the plant transformation may be mediated by, for example, Agrobacterium tumefaciens. In example embodiments, redifferentiation of the transformed plant from the transformed plant cell may be carried out using any related method in the art.
Embodiments relate to a transformed plant, which may be produced by the above-described method. The plant may preferably be, but is not limited to, a monocot plant, and more preferably may be rice, barley, wheat, maize, millet or Indian millet. Embodiments relate to a transformed seed. In embodiments, the transformed seed may be obtained from a transformed plant. A seed preferably may be derived from, but is not limited to, a monocot plant, and more preferably may be from rice, barley, wheat, maize, millet or Indian millet. Embodiments relate to a transformed cell. In embodiments, the transformed cell may be obtained from a transformed plant. A cell preferably may be derived from, but is not limited to, a monocot plant, and more preferably may be from rice, barley, wheat, maize, millet or Indian millet.
Embodiments relate to PCR primers. In embodiments, the primer may include SEQ ID NOS: 67 to 106. In embodiments, the primer may be for a constitutive expression promoter which may be configured to transform a plant, such as a monocot plant. In example embodiments, a promoter may react with at least one of SEQ ID NO: 1 through SEQ ID NO: 20. In example embodiments, a promoter may include a complementary base sequence to the at least one SEQ ID NO: 67 through SEQ ID NO: 106.
Embodiments relate to variants of a primer which may include a variant of at least one of SEQ ID NO: 67 through SEQ ID NO: 106. In embodiments, a variant primer may have different base sequences but include functional characteristics similar to those of at least one of SEQ ID NO: 67 through SEQ ID NO: 106. A variant may result from at least one of a substitution, deletion and insertion of nucleic acid base(s), or combinations thereof, including functional fragments thereof. In example embodiments, a variant of a primer may have a sequence identity of at least 70%, preferably at least 80%, even more preferably 90%, and most preferably at least 95% to the at least one of SEQ ID NO: 67 through SEQ ID NO: 106. In example embodiments, a primer may include a base sequence complementary to the variant at least one of SEQ ID NO: 67 through SEQ ID NO: 106.
Hereinafter, the present invention will be described in further detail with reference to example embodiments. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
1. Embodiment of Selection and Extraction of Promoter Sequences
Using the rice genome sequences of the international rice genome sequencing project (IRGSP), which was established in 1997 and completed the sequencing of the rice genome in December, 2004, and gene annotation data from the Institute for Genomic Research (TIGR) which carried out gene annotation based on the rice genome sequences, a region was selected to investigate its activity in a transformation, for example its activity in a vector. An annotated bacterial artificial chromosome (BAC) was selected, and about 2 kbp of sequence upstream from a ATG start codon of a coding sequence (CDS) was selected to be investigated as a promoter region. Only the 2-kbp sequence was extracted, separately, and used as a template for constructing PCR primers for isolating about 1.7-2-kb promoters from the 2-kbp sequence.
2. Embodiment of Analysis of a Constitutive Expression Gene by RT-PCR
For the analysis of a constitutive expression gene, samples were collected from seeds and leaf, root and flower tissues of 5-day-old (or 7-day-old), 20-day-old, 30-day-old and 60-day-old seedlings. For the preparation of the samples, the seeds were disinfected with about 70% ethanol and about 20% chlorax solutions, grown in a dark condition for about 5 days, and then developed in a greenhouse. To extract the total RNA, an RNeasy plant mini-kit (Qiagen, Cat. No. 74904) was used. A first-strand cDNA was synthesized using about 400 ng of the extracted total RNA (Invitrogen, Cat. No. 18080-051), and PCR was performed using about 1 μl of the synthesized cDNA product as a template. The primers used in the PCR reaction were as follows, and an ubiquitin (Ubi) primer set was used as a cDNA loading control.










Forward primer APX:




5′- GACCTCTAGACCGCCGTATT-3′
(SEQ ID NO: 21)



 



Reverse primer APX:




5′- GCCAACCACTCGCAATCCAA-3′
(SEQ ID NO: 22)



 



Forward primer SCP1:




5′- TCGCTGCCTACGCCAACATC-3′
(SEQ ID NO: 23)



 



Reverse primer SCP1:




5′- TCGCCGAACTAGCAGGTGAG-3′
(SEQ ID NO: 24)



 



Forward primer PGD1:




5′- CCGTGAGCTAGCGAGGATCT-3′
(SEQ ID NO: 25)



 



Reverse primer PGD1:




5′- CCGGTAGGAGTCGAAGTACG-3′
(SEQ ID NO: 26)



 



Forward primer R1G1:




5′- CTTCTCGATTGCCGTGTGCT-3′
(SEQ ID NO: 27)



 



Reverse primer R1G1:




5′- GCAAGTCTCAAGCTCTCAAT-3′
(SEQ ID NO: 28)



 



Forward primer EIF5:




5′- GATCTGCGCTCTGAAGGATA-3′
(SEQ ID NO: 29)



 



Reverse primer EIF5:




5′- AACCGCAAGATGGAACAACG-3′
(SEQ ID NO: 30)



 



Forward primer PF1:




5′- GGTCTCTTCGCCAAGCTCCT-3′
(SEQ ID NO: 31)



 



Reverse primer PF1:




5′- CGCCTCCTCCTTCTTCTCCT-3′
(SEQ ID NO: 32)



 



Forward primer PF2:




5′- AGCTCAAGGACCTTCAGTTC-3′
(SEQ ID NO: 33)



 



Reverse primer PF2:




5′- ACGGCGGACTGCATAGATAA-3′
(SEQ ID NO: 34)



 



Forward primer PF3:




5′- GGTCACTCCATCGTCAGAAT-3′
(SEQ ID NO: 35)



 



Reverse primer PF3:




5′- ACTTGCTCCACACTGATCAC-3′
(SEQ ID NO: 36)



 



Forward primer PF4:




5′- CAATGTGGCAGAGCTGATGG-3′
(SEQ ID NO: 37)



 



Reverse primer PF4:




5′- GGTCTGTAGGCACGACATAG-3′
(SEQ ID NO: 38)



 



Forward primer PF5:




5′- GGAGTCCTGCACTTACCATA-3′
(SEQ ID NO: 39)



 



Reverse primer PF5:




5′- CCATGGCGTACTTCTGTGTC-3′
(SEQ ID NO: 40)



 



Forward primer PF6:




5′- GAAGCTGTACGCCAAGGT-3′
(SEQ ID NO: 41)



 



Reverse primer PF6:




5′- TAGGTGCGAGCAACATTAGG-3′
(SEQ ID NO: 42)



 



Forward primer PF7:




5′- CACAGCCACACGAAGCCATA-3′
(SEQ ID NO: 43)



 



Reverse primer PF7:




5′- GCACAATGCCGATCGCAACA-3′
(SEQ ID NO: 44)



 



Forward primer SC1:




5′- AGCACCTTCCACCGCGTGAT-3′
(SEQ ID NO: 45)



 



Reverse primer SC1:




5′- TTCGCCATGGACAGGATGCC-3′
(SEQ ID NO: 46)



 



Forward primer SC2:




5′- CTGCGGAGGCATACCTTGTT-3′
(SEQ ID NO: 47)



 



Reverse primer SC2:




5′- ACACTACGACGCATGCTTCA-3′
(SEQ ID NO: 48)



 



Reverse primer SC3:




5′- AACGTGGACGGTGTTCATCA-3′
(SEQ ID NO: 49)



 



Reverse primer SC3:




5′- CAACTGCACTGGACGGCTTA-3′
(SEQ ID NO: 50)



 



Forward primer SC4:




5′- AACACCTTCGGCACCAGGAT-3′
(SEQ ID NO: 51)



 



Reverse primer SC4:




5′- AAGCGAACAGCAGCAGTCAG-3′
(SEQ ID NO: 52)



 



Forward primer SC5:




5′- CATCTTGCGGTCGGAGAA-3′
SEQ ID NO: 53)



 



Reverse primer SC5:




5′- TACGCATCCTCTGTGATGGT-3′
(SEQ ID NO: 54)



 



Forward primer SC6:




5′- CATTTGGTTCTGGCCCACCT-3′
(SEQ ID NO: 55)



 



Reverse primer SC6:




5′- GTCTGCCACCAGAGCTCCTA-3′
(SEQ ID NO: 56)



 



Forward primer SC7:




5′- CGTCACCAAGTTCACTTC-3′
(SEQ ID NO: 57)



 



Reverse primer SC7:




5′- CCACCTAATTCTTCTTACAGTC-3′
SEQ ID NO: 58)



 



Forward primer SC8:




5′- CAAGGCTGTGACCAAGTT-3′
(SEQ ID NO: 59)



 



Reverse primer SC8:




5′- CTACAGCACAGTACCATACC-3′
(SEQ ID NO: 60)



 



Forward primer OsCc1:




5′- ACTCTACGGCCAACAAGAAC-3′
(SAEQ ID NO: 61)



 



Reverse primer OsCc1:




5′- CTCCTGTGGCTTCTTCAACC-3′
(SEQ ID NO: 62)



 



Forward primer Act1:




5′- ATGGTGTCAGCCACACTGTC-3′
(SEQ ID NO: 63)



 



Reverse primer Actl:




5′- TAACCACGCTCCGTCAGGAT-3′
(SEQ ID NO: 64)



 



Forward primer OsUbi:




5′- ATGGAGCTGCTGCTGTTCTA-3′
(SEQ ID NO: 65)



 



Reverse primer OsUbi:




5′- TTCTTCCATGCTGCTCTACC-3′
(SEQ ID NO: 66)






The PCR reaction was performed in a PTC 200 PCR machine (MJ research) using about 1 μl of cDNA, 2× Taq premix (Solgent. Co. Cat. No. EP051020-T2B6-1), about 4 pmol of each template-specific primer in a total reaction volume of about 20 μl for about 32 cycles, each consisting of about 95° C. for about 30 sec, about 55° C. for about 30 sec and about 72° C. for about 1 min.
3. Embodiments of Amplification and Isolation of Promoters
Using the isolated 2-kbp promoter sequence as a template and a primer designer 4 program (ver. 4.20, Scientific & Educational software), PCR primers for isolating about 1.8-2-kb promoters were designed. The design conditions were as follows: the GC content of PCR primers: about 40-60%, Tm: about 55-65° C., and the concentrations of salt and free Mg: about 0 and about 0.15 mM, respectively. The primers (PCR primers) were designed such that the template-specific region was about 20 bp in length and the 5′ adaptor sequence was about 12 bp in length. The adaptor sequence was inserted for site-specific recombination other than existing cloning methods which are performed using restriction enzymes and DNA ligase. DNA used as a template was obtained by seeding a Japonica type Nipponbare cultivar rice, growing the plant in a greenhouse for about 3 weeks, cutting only the leap portion from the plant, and extracting genomic DNA from the leaf. The genomic DNA was obtained by freezing the cut leaf rapidly with liquid nitrogen, crushing the frozen leaf finely with a mortar and pestle, and then isolating the genomic DNA from the crushed leaf using DNAzol solution (molecular research center, Cat. No. DN128). The first reaction was carried out to isolate a specific promoter from the rice genome and performed using 32-bp template-specific primers linked with a 12-bp adaptor sequence. The primer sequences were as follows:









Forward template-specific primer:



5′-AAAAAGCAGGCT-tempate specific sequence-3′



 



Reverse template-specific primer:



5′-AGAAAGCTGGGT-template specific sequence-3′






Embodiments of gene-specific primer sequences were as follows:









a. APX promoter primers



(SEQ ID NO: 67)



Forward primer:



5′-AAAAAGCAGGCTgtaaggtgacatggcatatc-3′



 



(SEQ ID NO: 68)



Reverse primer:



5′-AGAAAGCTGGGTccaatccgaatcaatcaatc-3′



 



b. SCP1 promoter primers



(SEQ ID NO: 69)



Forward primer:



5′-AAAAAGCAGGCTttgactttttctgcgaagaa-3′



 



(SEQ ID NO: 70)



Reverse primer:



5′-AGAAAGCTGGGTtaactcttgccggaaaagaa-3′



 



c. PGD1 promoter primers



(SEQ ID NO: 71)



Forward primer:



5′-AAAAAGCAGGCTtagatatgccgaacatgacc-3′



 



(SEQ ID NO: 72)



Reverse primer:



5′-AGAAAGCTGGGTgcagatagatgcaccaaatg-3′



 



d. R1G1 promoter primers



(SEQ ID NO: 73)



Forward primer:



5′-AAAAAGCAGGCTatagctgttgtactgatgtc-3′



 



(SEQ ID NO: 74)



Reverse primer:



5′-AGAAAGCTGGGTtctctcgcagtattaccaac-3′



 



e. EIF-5A promoter primers



(SEQ ID NO: 75)



Forward primer:



5′-AAAAAGCAGGCTttgttccacctcatcattaa-3′



 



(SEQ ID NO: 76)



Reverse primer:



5′-AGAAAGCTGGGTcaacctgccaccaacaacaa-3′



 



f. PF1 promoter primers



(SEQ ID NO: 77)



Forward primer:



5′-AAAAAGCAGGCTctcggtgaagatagagaagg-3′



 



(SEQ ID NO: 78)



Reverse primer:



5′-AGAAAGCTGGGTctcgagctgatctacgaact-3′



 



g. PF2 promoter primers



(SEQ ID NO: 79)



Forward primer:



5′-AAAAAGCAGGCTcaacgggacactgtcgattc-3′



 



(SEQ ID NO: 80)



Reverse primer:



5′-AGAAAGCTGGGTggtgccgcctcttcaaatct-3′



 



h. PF3 promoter primers



(SEQ ID NO: 81)



Forward primer:



5′-AAAAAGCAGGCTtcgttccaacgtaccgcaat-3′



 



(SEQ ID NO: 82)



Reverse primer:



5′-AGAAAGCTGGGTgggagagctgagtgagcaga-3′



 



i. PF4 promoter primers



(SEQ ID NO: 83)



Forward primer:



5′-AAAAAGCAGGCTtctggcatcgatatgctcct-3′



 



(SEQ ID NO: 84)



Reverse primer:



5′-AGAAAGCTGGGTtggagtcacgcgagatacct-3′



 



j. PF5 promoter primers



(SEQ ID NO: 85)



Forward primer:



5′-AAAAAGCAGGCTgctgattccgtgcacctcat-3′



 



(SEQ ID NO: 86)



Reverse primer:



5′-AGAAAGCTGGGTgatcctcttggacgccatcg-3′



 



k. PF6 promoter primers



(SEQ ID NO: 87)



Forward primer:



5′-AAAAAGCAGGCTggaccaaccgaagtccttcc-3′



 



(SEQ ID NO: 88)



Reverse primer:



5′-AGAAAGCTGGGTtcctgcgcttgaaggtct-3′



 



l. PF7 promoter primers



(SEQ ID NO: 89)



Forward primer:



5′-AAAAAGCAGGCTaactgaacagggccttacca-3′



 



(SEQ ID NO: 90)



Reverse primer:



5′-AGAAAGCTGGGTcggcaacgaggtcgatac-3′



 



m. SC1 promoter primers



(SEQ ID NO: 91)



Forward primer:



5′-AAAAAGCAGGCTgatcacttgtggcagccata-3′



 



(SEQ ID NO: 92)



Reverse primer:



5′-AGAAAGCTGGGTcgctagggttttgcgaattt-3′



 



n. SC2 promoter primers



(SEQ ID NO: 93)



Forward primer:



5′-AAAAAGCAGGCTttacgtatagccttttcctt-3′



 



(SEQ ID NO: 94)



Reverse primer:



5′-AGAAAGCTGGGTgacagaatatgctgtgacaa-3′



 



o. SC3 promoter primers



(SEQ ID NO: 95)



Forward primer:



5′-AAAAAGCAGGCTaccgatgtagatgaatacttcc-3′



 



(SEQ ID NO: 96)



Reverse primer:



5′-AGAAAGCTGGGTatccttccgaaacctgaac-3′



 



p. SC4 promoter primers



(SEQ ID NO: 97)



Forward primer:



5′-AAAAAGCAGGCTgctcgtaggacaagcaggaa-3′



 



(SEQ ID NO: 98)



Reverse primer:



5′-AGAAAGCTGGGTcttgcgtgctcctgatctct-3′



 



q. SC5 promoter primers



(SEQ ID NO: 99)



Forward primer:



5′-AAAAAGCAGGCTtcctcttgccccttcctcgg-3′



 



(SEQ ID NO: 100)



Reverse primer:



5′-AGAAAGCTGGGTtgtgacgtggcagtctgaca-3′



 



r. SC6 promoter primers



(SEQ ID NO: 101)



Forward primer:



5′-AAAAAGCAGGCTgtgcactcaaggtcctatgg-3′



 



(SEQ ID NO: 102)



Reverse primer:



5′-AGAAAGCTGGGTtggcatacagcactccctct-3′



 



s. SC7 promoter primers



(SEQ ID NO: 103)



Forward primer:



5′-AAAAAGCAGGCTgtcgaactcaccgtgcacta-3′



 



(SEQ ID NO: 104)



Reverse primer:



5′-AGAAAGCTGGGTtggatgctgctctcttcttctc-3′



 



t. SC8 promoter primers



(SEQ ID NO: 105)



Forward primer:



5′-AAAAAGCAGGCTaaaaaacttgactgcccaaaa-3′



 



(SEQ ID NO: 106)



Reverse primer:



5′-AGAAAGCTGGGTggaaacggtggtggttct-3′






A first PCR reaction was carried out using about 50 ng of genomic DNA, 2× Taq premix (Solgent. Co. Cat. No. EP051020-T2B6-1) and about 10 pmol of each template-specific primer in a total reaction volume of about 50 μl for about 30 cycles, each consisting of about 95° C. for about 1 min, about 55° C. for about 1 min and about 68° C. for about 2 min.
A second PCR reaction was carried out to insert and amplify a specific adaptor sequence (att site) which may be used to insert a promoter into a transformation vector. The length of the sequence to be additionally inserted into the promoter was about 29 bp. To increase the efficiency of PCR, only a portion (12 bp) of the sequence was attached to the template-specific sequence by overhang and subjected to the first PCR reaction. Then, about 1/50 (1 μl) of the PCR reaction solution was taken and subjected to the second PCR reaction using primers (adaptor sequence primers) having full-length recombinant sequences. Thus, the PCR product had all the att sequences for recombination with the promoter. The adaptor primer sequences were as follows:









(SEQ ID NO: 107)



attB1 adaptor primer:



5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′



 



(SEQ ID NO: 108)



attB2 adaptor primer:



5′-GGGGACCACTTTGTACAAGAAAGCTGGGT-3′






The second PCR reaction was carried out using about 1 μl of the first PCR product, 2× Taq premix (Solgent. Co. Cat. No. EP051020-T2B6-1) and about 2 pmol of each adaptor primer in a total reaction volume of about 50 μl for about 5 cycles, each consisting of about 95° C. for about 30 sec, about 45° C. for about 30 sec and about 68° C. for about 2 min, followed by about 20 cycles, each consisting of about 95° C. for about 30 sec, about 55° C. for about 30 sec and about 68° C. for about 2 min. The PCR reactions were carried out using a Gateway system (Invitrogen, Cat. No. 12535-029) according to the method suggested by Invitrogen.
4. Embodiments of Cloning of Amplified Promoters
The promoter was inserted into a vector using a Gateway system (Invitrogen, Cat. No. 12535-029). The amplified promoter was electrophoresed on about 1% agrose gel, separated into bands on the gel and purified using the Mega-spin agarose gel extraction kit (Intron, Cat. No. 17183). A BP reaction was carried out using about 5 μl of the purified promoter, about 4 μl of a BP clonase enzyme mixture, about 4 μl of 5×BP reaction buffer, about 300 ng/2 μl of a pDONR vector, and TE buffer (about 10 mM Tris/pH about 8.0, about 1 mM EDTA) in a total reaction volume of about 20 μl at about 25° C. for about 16 hours. Then, about 6 μl of an LR clonase enzyme mixture, about 1 μl of about 0.75 M NaCl and about 450 ng/3 μl of a transformation vector were added to the reaction product and subjected to an LR reaction in a total reaction volume of about 30 μl at about 25° C. for about 8 hours. About 3 μl of proteinase was added thereto and allowed to react at about 37° C. for about 1 hour, and then about 2 μl of the reaction product was taken and transformed into DH5α competent cells. The transformed DH5α cells were plated in LB agar medium containing about 50 μg/ml of a Spectinomycin antibiotic and were grown in an incubator at about 37° C. for about 12 hours. DNA was extracted from the selected cells, and whether the promoter has been inserted into the extracted DNA was confirmed by PCR. Then, the DNA was subjected to sequencing and BLASTN analysis to confirm complete insertion of the isolated promoter.
A vector (pMJ401) is described as follows. Between the right-border sequence and the left-border sequence, a cassette to be replaced with a promoter according to embodiments subsequent to recombination is linked with the visible marker gene GFP and a protease inhibitor II at the 3′ end. The cassette has the att sequences to facilitate BP and LR reactions. The selection gene (selection marker gene) was prepared such that the herbicide-resistant gene bar (phosphinotricine acetyltransferase gene) was controlled by the constitutive expression promoter OsCc1 (see U.S. Pat. No. 6,958,434). The gene was linked with a nopalin synthase (NOS) terminator. Also, a MAR sequence was attached to the terminal of the right-border sequence to minimize the change in expression in various sites of the chromosome, such that only the inherent activity of the promoter could be measured.
5. Embodiments of Agrobacterium-Mediated Transformation of Rice
About 70% (v/v) ethanol was added to TO hulled rice seeds (Oryza sativa L. cv Nakdong) and gently mixed for about 1 minute to wash the seeds. The washed seeds were sterilized by treatment in about 20% chlorax for about 1 hour and washed several times with sterile water. For transformation, the washed rice seeds were incubated on a callus induction medium (2N6) for about one month according to the method of Jang et al (Jang, I-C. et al., Mol breeding, 5:453-461, 1999) to induce embryonic callus, and then were co-cultivated with Agrobacterium obtained by an Agrobacterium triple mating method so as to insert the promoter-inserted transformation vector into the rice genome. Then, the plant was incubated on a 2N6-CP medium for selecting transformed callus for about one month. The grown cells were selected and cultured in a redifferentiation medium (MS-CP) for about 1-2 months, and a redifferentiated plant was acclimated in a greenhouse. The acclimated T0 rice was treated with the non-selective herbicide basta, and only the plants showing herbicide resistance were selected and subjected to a progeny test.
6. Embodiments of Analysis of Promoter Activity by RT-PCR and Real Time qRT-PCR
For the analysis of promoter activity, total RNA was extracted from the seeds of transformed plants and the leaf, root and flower tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old seedlings. An RNeasy plant mini-kit (Qiagen, Cat. No. 74904) was used to extract the total RNA from each tissue. A first-strand cDNA was synthesized using about 400 ng of the extracted total RNA (Invitrogen, Cat. No. 18080-051), and PCR was performed using about 1 μl of the synthesized cDNA product as a template. The PCR reaction was performed using two kinds of primer sets. The first primer set was a primer set (primer GFP) for comparing the expression levels of GFP inserted downstream of the promoters, and the second primer set was a primer set (primer Ubi) as a cDNA loading control. The primer sequences were as follows.








Forward primer GFP:



5′-CAGCACGACTTCTTCAAGTCC-3′
(SEQ ID NO: 109)


 


Reverse primer GFP:



5′-CTTCAGCTCGATGCGGTTCAC-3′
(SEQ ID NO: 110)


 


Forward primer OsUbi:



5′-ATGGAGCTGCTGCTGTTCTA-3′
(SEQ ID NO: 65)


 


Reverse primer OsUbi:



5′-TTCTTCCATGCTGCTCTACC-3′
(SEQ ID NO: 66)






The RT-PCR reaction was carried out in a PTC 200 PCR machine (MJ research) using about 1 μl of cDNA, 2× Taq premix (Solgent. Co. Cat. No. EP051020-T2B6-1) and about 2 pmol of each template-specific primer in a total reaction volume of about 20 μl for about 39 cycles, each consisting of about 95° C. for about 30 sec, about 55° C. for about 30 sec and about 72° C. for about 30 sec.
The real-time qRT-PCR reaction was carried out in Mx3000P (Stratagene) using about 1 μl of cDNA, 2×SYBR green qRT-PCR premix (Invitrogen. Cat. No. 11765-100) and about 2 pmol of each template-specific primer in a total reaction volume of about 20 μl for about 40 cycles, each consisting of about 95° C. for about 15 sec and about 60° C. for about 30 sec. After completion of the reaction, the promoter activity was quantitatively analyzed using the program Mx3000P (Stratagene) according to the manufacturer's instruction.
7. Embodiments of Observation of GFP Expression and Analysis of Promoter Activity in Each Organ of Rice
For the analysis of promoter activity, the expression of fluorescence of the marker gene GFP was observed in seeds and the leaf, root and flower organs of seedlings which were about 1 month old after germination. The observation of gene expression was carried out from the T2 generation in which the insertion and separation of the gene could be reliably observed. The observation of GPF expression in seeds was carried out by hulling the seeds and observing GFP expression in the embryo and endosperm of the hulled seeds using an LAS3000 system (Fuji Photo Film Co., Ltd.) and a SZX9-3122 stereomicroscope (Olympus, Tokyo, Japan). Meanwhile, in order to observe GFP expression in seedlings which were about 1 month old after germination, seeds in which GFP expression has been observed were selected and sterilized in ethanol and about 20% chlorax solution. Then, to examine the activity of the selection marker “bar” gene, the selected seeds were germinated in a PPT-containing MS-P medium (about 4 mg/l PPT) in a dark condition for about 3 days, and then incubated in a light condition for about 2 days. The germinated etiolated seedlings were grown in soil for about 25 days, and then the expression of the gene in the leaf and root of the seedlings were observed using an LAS3000 system under the following conditions: precision, standard, exposure time: about 1 sec (excitation filter: about 460 nm, and barrier filter: about 510 nm). Flowers were collected before opening from the seedlings and observed using a SZX9-3122 stereomicroscope (Olympus, Tokyo, Japan) before and after removing the glume. In this way, GFP fluorescence in each organ in rice was observed.
EXAMPLE 1
Analysis of Expression of Genes in Each Rice Tissue
To examine the tissue-specific activities of the APX, SCP1, PGD1, R1G1 and EIF-5A promoters, samples were collected from the seeds of the transformed rice and the leaf (L in FIG. 1), root (R in FIG. 1) and flower tissues of 5-day-old (or 7-day-old), 20-day-old, 30-day-old and 60-day-old seedlings, and total RNA was extracted from each sample. cDNA was synthesized using the RNA as a template and amplified by PCR. The PCR products were electrophoresed on about 2% agarose gel.
FIG. 1 shows the results obtained by comparing the expression patterns of twenty constitutive expression genes in various tissues of rice using RT-PCR. As can be seen in FIG. 1, the APX, SCP1, PGD1, R1G1, EIF-5A (indicated as “EIF5” as the figure; hereinafter the same), PF1, PF2, PF3, PF4, PF5, PF6, PF7, SC1, SC2, SC3, SC4, SC5, SC6, SC7 and SC8 genes used were expressed substantially uniformly in various tissues of rice. Also, these genes showed expression patterns similar to those of related constitutive expression genes OsCc1 and Act1, suggesting they may be constitutive expression genes.
EXAMPLE 2
Construction of Rice Transformation Vector and Structure of Promoter
A vector for analyzing promoter activity was constructed and is shown in FIG. 2. FIG. 2 shows a pMJ401 vector. The vector may be a parent vector for cloning the isolated promoter by PCR. The attR1 and attR2 sites are sites where recombination (site-specific recombination) with the attL1 and attL2 sequences of the promoter may occur after a BP reaction. After an LR reaction, a cassette was replaced with the promoter and the attR1 and attR2 sequences were also replaced with attB1 and attB2 sequences.
A description is provided as follows: MAR: matrix attachment region (1.3 kb), X98408; cassette B: conversion cassette B (1.7 kb; Invitrogen, Cat. No. 11828-019); GFP: modified green fluorescent protein gene (0.74 kb), U84737; TPINII: protease inhibitor II terminator (1.0 kb), X04118; OsCc1: cytochrome c promoter (0.92 kb), Af399666; BAR: phosphinotricine acetyltransferase gene (0.59 kb), X17220; and TNOS: nopaline synthase terminator (0.28 kb). FIG. 3 shows the structures of embodiments of promoters disposed in a rice genome.
EXAMPLE 3
Analysis of Promoter Activity (e.g., GFP Expression Level) in Each Tissue of Transformed Rice by RT-PCR
RNA was extracted from the seeds of the transformed rice and the leaf, root and flower tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old seedlings. cDNA was synthesized using the RNA as a template and amplified by PCR. The PCR products were electrophoresed on about 2% agarose gel. Each PCR product was loaded in an amount of about 5 μl. FIG. 4 shows the results obtained by semi-quantitatively analyzing the GFP expression level caused by each promoter in the rice seeds and the leaf (L), root (R) and flower tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old seedlings using RT-PCR. GFP was a PCR product amplified with GFP primers and was used to compare the expression levels of the GFP gene inserted downstream of the promoters. The PCR product was 141 bp in size. In view of the difference in gene expression according to the variation between events, the analysis of the transformants for each promoter was carried out using 3 events having different promoter insertion sites.
FIG. 4 illustrated that, for example, APX, SCP1, PGD1, R1G1 and EIF5 promoters were novel promoters, which showed gene expression levels similar to or lower than those of the related OsCc1 promoter, Act1 promoter and maize Ubi1 promoter (ZmUbi), but induced gene expression substantially uniformly in substantially all the tissues.
EXAMPLE 4
Analysis of Promoter Activity in Each Rice Tissue by Real Time qRT-PCR
FIG. 5 and FIG. 6 show the results obtained by quantitatively analyzing the expression pattern of the GFP gene according to the activity of each promoter in each tissue of the transformed rice. Similar to in FIG. 4, RNA was extracted from the seeds of the transformed rice and the leaf, root and flower tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old seedlings. cDNA was synthesized using RNA as a template and amplified by PCR. Then, the PCR product was subjected to real time qRT-PCR analysis using gene-specific primers of GFP used as a target gene. In view of the difference in gene expression according to the variation between events, the analysis of the transformants for each promoter was carried out using 3 events having different promoter insertion sites.
FIG. 5 quantitatively shows the activities of the promoters in each of the leaf and root tissues of 5-day-old, 20-day-old, 30-day-old and 60-day-old seedlings. FIG. 6 quantitatively shows the activities of the promoters in the seed, which is a reproductive and storage organ, and in the flower, which is another reproductive organ.
EXAMPLE 5
Comparison of Promoter Activity Between Transformants Having Different Insertion Sites
Because the activity of the promoter can greatly change according to its insertion location in the genome, such as the rice genome, the activity range predictable when the promoter is used to express a transgene may be determined by measuring the change in the activity of the promoter in a number of different transformants. Transformants (3-8 events per promoter) having different promoter insertion sites were grown for about 20 days in a greenhouse after germination, and then total RNA was extracted from the leaf and root tissues. cDNA was synthesized using the RNA as a template and amplified by PCR. Then, the PCR products were subjected to real time qRT-PCR analysis using gene-specific primers of GFP used as a target gene.
FIG. 7 shows the results obtained by quantitatively analyzing the expression pattern of the GFP gene according to the activity of each promoter in the transformed rice. The obtained distribution of change in promoter activity for each transformed event may be a good index for predicting the minimum and maximum values of expression level when using the corresponding promoter for expression of a transgene.
EXAMPLE 6
Observation of GFP Fluorescence in Leaf and Root Tissues of Rice Transformed with Promoter
To analyze the activities of the promoters according to embodiments, the leaf and root tissues of rice plants transformed with promoters, which were about 1 month old after germination, and GFP fluorescence in the tissues, were observed using a SZX9-3122 stereomicroscope SZX9-3122 (Olympus, Tokyo, Japan).
FIG. 8 shows the expression of GFP fluorescence in the leaf and root tissues of rice plants transformed with promoters. The description of each gene shown in FIG. 8 is as follows: NC: negative control Oryza sativa L. cv Nakdong (non-transformed rice); ZmUbi: maize Ubi1 promoter; Act1: rice Actin1 promoter; and OsCc1: rice cytochrome c promoter.
In the leaf and root tissue of the negative control, the expression of GFP fluorescence was not observed, whereas in the leaf and root tissues of the rice plants transformed with the promoters according to embodiments, the expression of GFP fluorescence was clearly and substantially uniformly observed. This visually shows that the activities of the promoters according to embodiments were distributed substantially uniformly in the leaf and root tissues of the transformed rice plants in the same manner or better as in the case of the rice plants transformed with each of the positive controls, namely ZmUbi, Actin1 and OsCc1.
EXAMPLE 7
Observation of GFP Fluorescence in Reproductive Organ of Rice Transformed with Promoter
In order to analyze the activities of promoters according to embodiments in the reproductive organ of rice, the hulled seeds of the rice plant transformed with each of the promoters were collected, and GFP fluorescence in the hulled seeds was observed using a SZX9-3122 stereomicroscope (Olympus, Tokyo, Japan). The T2 generation seeds showing homozygous GFP expression therein were sowed in a greenhouse before opening, and GFP fluorescence in the collected seeds was observed using a SZX9-3122 stereomicroscope (Olympus, Tokyo, Japan) before and after removing the glume.
FIG. 9 and FIG. 10 show the expression of GFP fluorescence in the seed and flower of the transformed rice. The description of each gene shown in FIG. 9 and FIG. 10 is as follows. NC: negative control Oryza sativa L. cv Nakdong (non-transformed rice); ZmUbi: maize Ubi1 promoter; Act1: rice Actin1 promoter; and OsCc1: rice cytochrome c promoter.
For the rice plant transformed with the OsCc1 promoter, GFP fluorescence was observed in the rice seed, the leaf and root of the young seedling, and throughout the flower. For the rice plant transformed with the Act1 promoter, GFP fluorescence was observed not in the leaf and root of the rice plant, and also in the stamen. For the rice plant transformed with the ZmUbi promoter, GFP fluorescence was observed uniformly in all the flower organs, including lemma, palea, pistil and anther. For the APX, SCP1 and EIF5 promoters, GFP fluorescence was observed in the seed embryo in a manner similar to the case of OsCc1 and Act1. For the PGD1 and R1G1 promoters, GFP fluorescence was observed in substantially all the embryo and endosperm in a manner similar to the case of ZmUbi. For the SCP1, R1G1 and EIF5 promoters, the substantially uniform expression of GFP fluorescence was observed in the flower of the transformed rice. In particular for the APX and PGD1 promoters, outstanding GFP fluorescence was observed in the flower anther and ovary. For the illustrated five expression promoters in FIG. 9 and FIG. 10 according to embodiments, GFP fluorescence was observed substantially uniformly in substantially all the tissues of the rice plants, including seeds and flowers.
Notably, the results of expression of GFP fluorescence in each tissue of the rice plants transformed with promoters were partially different for the results of real time-qPCR. This may be a result of sensitivity differences between the analysis methods. Accordingly, the results of observation of GFP fluorescence may preferably be used as qualitative information.
It will be obvious and apparent to those skilled in the art that various modifications and variations can be made in the embodiments disclosed. Thus, it is intended that the disclosed embodiments cover the obvious and apparent modifications and variations, provided that they are within the scope of the appended claims and their equivalents.

## Claims

1. A promoter consisting of:
SEQ ID NO: 3.

2. The promoter of claim 1, wherein the promoter is a constitutive expression promoter operatively linked to a gene to transform a plant.

3. The promoter of claim 2, wherein the plant is a monocot plant comprising at least one of a rice, barley, wheat, maize, millet and Indian millet.

4. The promoter of claim 1, wherein the promoter expresses a gene substantially uniformly in substantially all organs and/or tissues of a plant.

5. The promoter of claim 1, comprising a base sequence complementary to the entire length of SEQ ID NO: 3.

6. A vector comprising:
a promoter consisting of SEQ ID NO: 3.

7. The vector of claim 6, wherein:
the vector is a recombinant plant expression vector; and
wherein the promoter is a constitutive expression promoter.

8. The vector of claim 7, wherein a target gene downstream of the promoter encoding a target protein is operably linked.

9. The vector of claim 6, wherein the promoter expresses a gene substantially uniformly in substantially all organs and/or tissues of a plant.

10. The vector of claim 6, wherein SEQ ID NO: 3 is a variant having a sequence identity of at least 95%.

11. A composition comprising at least one of:
a transformed plant comprising a promoter consisting of SEQ ID NO: 3;
a transformed seed comprising a promoter consisting of SEQ ID NO: 3; and
a transformed cell comprising a promoter consisting of SEQ ID NO: 3.

12. A method comprising:
forming a target protein by transforming a plant using a vector comprising a promoter consisting of SEQ ID NO: 3.

13. The method of claim 12, wherein the target protein comprises at least one of interleukin, interferon, platelet-derived growth factor, hemoglobin, elastin, collagen, insulin, fibroblast growth factor, human growth factor, human serum albumin and erythropoietin.

14. A method comprising at least one of:
transforming a plant cell with a vector comprising a promoter consisting of SEQ ID NO: 3; and
redifferentiating a transformed plant from the transformed plant cell.