KR20150092529A - funiculus specific expression promoter - Google Patents

Abstract

The present invention relates to a promoter, which specifically induces an expression to a funiculus tissue in a plant, a vector including the promoter, a transformant including the vector, and a transforming method. According to the present invention, since the promoter only specifically induces the expression in the funiculus tissue, the promoter can induce a gene expression in the funiculus tissue, and can be very usefully used to develop a high value-added new variety having an excellent quality or an effect of an increase in yield by controlling desorption from an end of a seed.

Description

A host-specific expression promoter (funiculus specific expression promoter)

The present invention relates to a promoter which induces expression specifically in a funiculus tissue in a plant, a vector containing the promoter, and a transformant comprising the vector.

Crop molecular breeding can use all kinds of genes as a material and it is possible to control the effect of breeding infinitely by dragging the breeding technique which was possible only by the existing genome unit as a gene unit, Technology. The cultivation area and the cultivation amount of genetically modified crops using this crop molecular breeding technique are continuously increasing all over the world, and the global market size of the commercialized genetically modified new person is from $ 4 billion in 2002 to $ 10 billion in 2010 And 15% of the total global seed market, which is 15% of the $ 30 billion market. Soybean, corn, cotton, and rapeseed are the major markets. In order to maximize the effect of genetically modified crops, the expression of exogenously inserted genes must be finely regulated by the developmental stage and tissue of the plant, and various promoters should be developed for this purpose.

Since the early 1980s, research has been conducted on promoters that regulate the expression of plant genes. It has been suggested that the promoter of cauliflower mosaic virus will induce strong expression in whole plant tissues, the nucleotide sequence of the promoter is revealed, and strong expression has been demonstrated in the plant body. The CaMV 35S (patent number: JP1993192172-A1) promoter has since become the universal promoter most commonly used in plants.

On the other hand, studies on the expression of a tissue-specific promoter that induces specific tissue-specific expression of plants have been steadily conducted, and a root tissue specific promoter (Elmayan and Tepfer, 1995, Transgenic Research 4: 388-396; Xu et al., 1995 , Plant Molecular Biology , 27: 237-248; Nitz et al., 2001, Plant Sci. 161: 337-346; Vaughan et al., 2006, Journal of Experimental Botany 57: 3901-3910; Vijaybhaskar et al., 2008 , Journal of Biosciences , 33: 185-193; Jeong et al., 2010, Plant Physiology , 153: 185-197; Noh et al., 2012, Transgenic Research 21: 265-278), storage root specific promoters al, 2012, Planta, 236: 1955-1965; Noh et al, 2012, Transgenic Research 21:..... 265-278), flower tissue specific promoter (van der Meer et al, 1990 , Plant Mol Biol 15: Plant Molecular Biology , 66: 379-388; Verdonk et al., 2008, Plant Biotechnology Journal , pp . 95-109; Ruiz-Rivero and Prat, 1998; Plant Mol. Biol. 36: 639-648; 6: 694-701; Liu et al., 2011, Pla nt Cell Reports 30: 2187-2194), approximately (anther) specific promoters (van Tunen et al, 1990, Plant Cell 2: 393-401; Kato et al, 2010, Plant Molecular Biology Reports 28:.. 381-387) , Seed-specific promoters (Bustos et al., 1989, Plant Cell 1: 839-853; Kridl et al., 1991, Seed Sci Res , 1: 209-219; Washida et al., 1999, Welham and Domoney, 2000, Plant Sci. 159: 289-299; Plant Mol. Biol. 40: 1-12; Furtado et al., 2008, Plant Biotechnology Journal 6: 679-693; Chung et al., 2008, Plant Cell Reports , 27: 29-37).

The main stem of the seed has been shown to regulate the maturation of the Arabidopsis seeds by the glutathione synthesis-related gene expressed in the carcass tissue as a tissue that connects the seed and seed pods (Cairns et al., 2006, Plant Physiology , 141: 446-455) and showed that seed mutagenesis did not occur when mutation occurred in the Def (Development funiculus) gene expressed in the host strain of soybean (Ayeh et al., 2009, BMC Plant Biology , 9: 76-82) . On the other hand, seeds harvested with pods such as beans have a problem that seed harvesting amount is significantly reduced when pods are opened before harvesting of seeds and seeds are removed from pods. In order to reproduce plants in a natural state, seeds There is a need to be easily removed from the pod. Therefore, there is a need to develop a technique for regulating the degree to which seeds are eliminated from the pod. For this purpose, there is a demand for the development of a promoter that induces expression specifically in the tissue of the main stem, which is a tissue connecting the seed and pod I have been there continuously.

KR 10-2007-0115325A

It is an object of the present invention to provide a promoter that induces a specific expression of a plant's funiculus tissue.

Another object of the present invention is to provide an expression vector comprising the promoter, a transformant comprising the vector, and a composition for transformation.

It is still another object of the present invention to provide a primer set capable of amplifying the promoter.

In order to achieve the above object, the inventors of the present invention cloned a promoter region of At5g55750 gene expressing seed-specific expression using Arabidopsis thaliana TAIR / ATTED DB to express a plant-specific expression of funiculus that connects seeds of seeds and seed pods And the activity of the promoter was confirmed in a transformant using Arabidopsis thaliana, thereby completing the present invention.

Specifically, the present invention comprises a base sequence represented by SEQ. ID. NO. 1, and provides a promoter which induces a specific expression of a main pathogenic tissue of a plant.

The present invention also provides an expression vector comprising the promoter.

The invention also provides an expression vector comprising the promoter and a gene operably linked thereto.

The present invention also provides a transformant comprising the promoter or the vector.

In addition, the present invention provides a composition for transforming a plant comprising any one of the promoter and the vector.

Also, the present invention provides a primer set comprising a forward primer consisting of SEQ ID NO: 2 and a reverse primer consisting of SEQ ID NO: 3 for amplifying a DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a promoter comprising a nucleotide sequence represented by SEQ ID NO: 1 and inducing a main pathogen-specific expression of a plant.

The promoter is a promoter capable of initiating transcription, and is active in a specific tissue, particularly a main pathology tissue. In addition, variants of the promoter sequence may be included within the scope of the present invention. The mutant includes all the nucleotide sequences having a functional characteristic similar to the nucleotide sequence of SEQ ID NO: 1 although the nucleotide sequence is changed. Specifically, the promoter may include a nucleotide sequence having 70% or more, 80% or more, 90% or more, or 95% or more homology with the nucleotide sequence of SEQ ID NO:

The percent of sequence homology to the polynucleotide is determined by comparing the comparison region with the two optimally aligned sequences, and some of the polynucleotide sequences in the comparison region are referenced (addition or deletion) to the optimal alignment of the two sequences , But may include additions or deletions (gaps).

The funiculus is a part of the bag attached to the epidermis of the plant's umbilical cord, and is called a seed disease when it matures. In the case of using the promoter of the present invention, the expression of the gene is induced in the host tissue, and the expression of the gene in the host tissue is induced It is possible to control the desorption of the seeds.

The promoter has the nucleotide sequence of SEQ ID NO: 1, and the nucleotide sequence is derived from -1 to -1200 from the translation initiation site of the coding sequence of the Arabidopsis At5g55750 gene. The promoter may induce the expression of the target gene in the host tissue of the plant seed.

The present invention also relates to an expression vector comprising said promoter and an expression vector comprising said promoter and a gene operably linked thereto.

The vector may preferably be a plant expression vector, and may include all kinds of vectors conventionally used as a plant expression vector.

The promoter in the expression vector may be located in front of the operably linked gene. Operably linked means that the recombinant DNA comprises a recombinant DNA comprising a suitable nucleic acid sequence necessary for the expression of the coding sequence by the gene and may for example be an expression cassette component functioning as a unit for expressing the heterologous protein.

The gene may be any gene that desires expression in a funiculus tissue, and the gene may be an exotic gene. For example, it may be a gene that encodes a protein capable of achieving a purpose useful for seed maturation and desorption control, such as a protein that regulates the desorption of a host disease, and a protein that regulates nutrient supply required for seed development.

The gene is located behind the promoter region in the host-specific expression vector, and may be expressed by fusion with the reporter gene, if necessary.

In one embodiment of the present invention, At5g55750: GUS in which the promoter of the present invention is inserted into a binary vector (pBI101) containing the GUS reporter gene was prepared, and the GUS reporter gene could be replaced with a foreign gene of another useful purpose have.

The present invention also relates to a transformant comprising the promoter or the vector and a composition for transforming the plant comprising the promoter or the vector.

The transformant may be a transformed plant transformed with the expression vector of the present invention. The plant is not limited to an expression cassette containing a main pathogen-specific promoter or a plant capable of transformation by insertion of an expression vector. The plant may be any one selected from the group consisting of the plant itself, the tissue, the cell, and the seed.

The plant cell may be any plant cell, and may be any of those selected from the group consisting of cultured cells, cultured tissues, culture media, and combinations thereof.

The plant tissue may be an undifferentiated or differentiated plant tissue. Examples of the plant tissue include roots, stems, leaves, pollen, seeds, cancer tissues, and various types of cells used for culturing. Examples thereof include single cells, protoplasts, And callus. In addition, the plant tissue may be in planta, or may be an organ culture, a tissue culture, or a cell culture.

The plant may be any one selected from the group consisting of food crops, vegetable crops, special crops, fruit trees, flowers, feed crops, and combinations thereof. The plant may preferably be a dicotyledonous plant.

The transfection composition may preferably comprise a culture of a microorganism comprising the expression vector of the present invention. The culture is a culture product obtained by culturing a microorganism comprising the vector of the present invention in a culture medium or a culture medium, and may include any one selected from the group consisting of an upper layer, a concentrate, a dried product and a combination thereof of the culture . And is not limited to the formulation, but may be liquid or solid.

In addition to the promoter or expression vector of the present invention, the transforming composition may further include additional components used for transforming plants. For example, the composition may include adjuvants, excipients, carriers, etc. to improve transformation efficiency .

The method of inserting the promoter of the present invention or an expression vector containing the promoter of the present invention into a plant may be a method commonly used in the technical field of the present invention. For example, the method of Agrobacterium method, osmotic pressure method, electroporation method, Methods, and combinations thereof, but the present invention is not limited thereto. Preferably, the Agrobacterium method can be used.

The transformant transformed by the transforming method of the present invention can express the gene specifically in the host tissue by the promoter of the present invention which is specifically expressed in the main pathology tissue, Can be regulated to improve the yield of the plant.

The present invention also provides a primer set comprising a forward primer consisting of SEQ ID NO: 2 and a reverse primer consisting of SEQ ID NO: 3 for amplifying a DNA fragment comprising the nucleotide sequence shown in SEQ ID NO: 1 and a plant set comprising the primer set To a screening kit.

The DNA fragment may be represented by SEQ ID NO: 1, and the nucleotide sequence of SEQ ID NO: 1 is derived from the Arabidopsis At5g55750 gene.

The primer set comprising SEQ ID NOS: 2 and 3 can amplify the host-specific promoter of the present invention.

The present invention also relates to a method for producing a target protein in a plant, wherein the target protein is produced in the plant main pathogens using the plant transformant.

The Arabidopsis specific promoter derived from Arabidopsis thaliana of the present invention induces gene expression in the funiculus tissue of the plant to regulate seed maturation and nutrient supply or to regulate desorption from seeds and seed pods, It can be very useful for the cultivation of new variety of high value-added seed having an increasing effect.

FIG. 1 is a diagram showing the results of tissue expression expression by RT-PCR of the At5g55750 gene, whose expression is expected to be regulated by the promoter of the present invention.
2 is a diagram showing a PCR result for promoter cloning for the present invention.
3 is a pBI101 vector schematic diagram of an expression vector used for promoter cloning according to the present invention.
FIG. 4A is a graph showing the result of analysis of the expression pattern of GUS by the regulation of the main disease tissue-specific promoter according to the present invention, and FIG. 4B is a photograph depicting the seeds on the left side of the pod and the right side shows seeds having a funiculus tissue .
FIG. 4B shows the results of analysis of internal expression patterns of GUS in the stage of maturation by controlling the At5g55750 promoter according to the present invention. From the left side, from the left side, the initial formation of the embryo to the immature seed, Fig.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

Example 1: Selection and confirmation of gene (At5g55750) specifically expressed in seeds

In order to obtain a gene specifically expressed in seeds, genes selected only in seeds based on the results of affymetrix 25k (ATHI) array disclosed in TAIR / ATTED DB were selected, Genes that were unable to identify the expression pattern were selected. Among them, genes located at At5g55750 were selected.

RT-PCR was performed to determine whether expression of the At5g55750 gene, which is presumed to express seed-specific expression, is actually expressed only in seeds. Each of TRI REAGENT ^® using the ^{(TRI REAGENT ® -RNA / DNA /} PROTEIN ISOLATION REAGENT, MOLECULAR RESEARCH CENTER, INC.), 5 main Arabidopsis (Arabidopsis thalina Columbia ecotype) to synthesize the standard cDNA used for RT-PCR Extraction of total RNA (total RNA) from tissues, flowers, inflorescence, leaves, roots, immature seeds and mature seeds of 7 major Arabidopsis thaliana Respectively.

5 μg of the total RNA (total RNA), 2.5 μM anchored-oligo (dT) ₁₈ primer, 1 × (8 mM MgCl ₂ ) transcriptor reverse transcriptase reaction buffer, 20 U protector ribonuclease inhibitor (protector RNase inhibitor), 1 mM deoxynucleotide mix, and 10 U transcriptor reverse transcriptase were mixed to a total volume of 20 μl and treated at 55 ° C for 70 minutes. And then treated at 85 ° C for 5 minutes to inactivate the transcriptor reverse transcriptase. A cDNA pool for each tissue was constructed as described above, and this cDNA pool was used as a template for RT-PCR.

Primers 1 and 2 (SEQ ID NOS: 4 and 5 in Table 1) were synthesized in the protein coding region based on the nucleotide sequence of the Tair site. Gene Amp ^® PCR System 2700 (Perkin Elmer, USA ) For 30 minutes at 95 ° C for 20 seconds, 60 ° C for 20 seconds, and 72 ° C for 30 seconds after the initial denaturation at 95 ° C for 5 minutes.

For RT-PCR
primer 5 '- GTAAACCCCGTCTTCAACG-3' SEQ ID NO: 4 5 '- CGCCAATAGTACCGTAAGGATAA-3' SEQ ID NO: 5

EF1a was used as an internal control and the transcription amount of EF1a in the tissue pool was almost the same. The results of RT-PCR using the At5g55750 gene specific primer are shown in FIG. As shown in Fig. 1, it was confirmed that At5g55750 gene is not expressed in flowers, leaves, stems, and roots, and is weakly expressed in immature seeds and strongly expressed in mature seeds.

Example 2: Promoter cloning of the Arabidopsis At5g55750 gene

The region deduced as the promoter of the gene was estimated as a base pair of 1200 bases. First extracting genomic DNA (gDNA) of Arabidopsis thaliana wild, and the 5 'end is the HindIII 3' end has included a BamHI and a synthetic primer represented by SEQ ID NO: 2 and SEQ ID NO: 3 in (Table 2).

Primers for promoter cloning 5 '- GGCGAAGCTTAAAAAAGCAGAGACTTGGACAG-3' SEQ ID NO: 2 5 '- ATTTGGATCCTCATGGTGCTGGGTGAGT-3' SEQ ID NO: 3

Using the gDNA as a template, the promoter region was amplified using primers of SEQ ID NOS: 2 and 3 and Phusion High-Fidelity DNA Polymerase (Phusion ^? DNA Polymerase, Thermo Scientific), a DNA polymerase having a proofreading function. The PCR reaction was carried out using Gene Amp ^® PCR System 2700 (Perkin Elmer, USA) for 30 min at 98 ° C for 10 min, 98 ° C for 10 sec, 58 ° C for 30 sec and 72 ° C for 40 sec. Respectively.

The result of PCR was confirmed on the agarose gel with 1220 (including the restriction enzyme sequence) base pair. The PCR product was terminated with HindIII and BamHI restriction enzymes, and HindIII of the pBI101 vector having the GUS reporter gene BamHI restriction enzyme recognition site. Thereafter, E. coli colony-PCR was performed to confirm that the PCR product was inserted, and one of the clones was sequenced to confirm that the correct nucleotide sequence was cloned (see SEQ ID NO: 1). Therefore, the above site was referred to as At5g55750 promoter, and the following transfection experiments were carried out.

Example 3: Arabidopsis transformation of the At5g55750 promoter

The pBI101 vector used for the At5g55750 promoter cloning is a binary vector having a GUS (β-glucuronidase) reporter gene between the left board and the right board and capable of transforming into plants 3).

This vector was transformed into Agrobacterium (GV3101) and transformed into Arabidopsis by the method of flower dipping (Clougher al., Plant J., 16 (6): 735-743, 1998) Respectively. Transgenic Arabidopsis thaliana was selected on a medium containing the antibiotic kanamycin.

Example 4: Histochemical staining analysis of transgenic Arabidopsis seeds

GUS activity from seeds of selected transgenic plants was investigated using histochemical staining. The seeds were seeded with 1 mM X-Gluc (5-bromo-4-chloro 3-indoly-β-glucuronidase), 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM ferricyanide , Potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% Triton X-100 at 37 ° C for 24 hours. After removing the solution, ethanol and acetic acid were dissolved in 9 : 1 at 25 ° C for 24 hours, followed by washing with 90% ethanol for 30 minutes. The procedure was repeated twice. Then, the pigment was removed secondarily for 24 hours at 25 ° C in a solution containing 2.5 g of chloral hydrate in 1 ml of 30% glycerol, followed by microscopic observation using an optical microscope (Olympus model BX-51 ), The activity of GUS was observed, and the results are shown in Fig.

As shown in Fig. 4, GUS staining was specifically confirmed in the seedling's funiculus connecting seed and seed pod (see Figs. 4A and 4B). In addition, GUS staining was observed only in the seed funiculus during development of the seed embryo, and GUS staining was not observed in other parts of the cotyledon even in cotyledon seeds (Fig. 4b) ). These results suggest that the At5g55750 promoter has a strong specific promoter activity in the seed host (funiculus).

<110> Korea University Research and Business Foundation <120> FUNICULUS SPECIFIC EXPRESSION PROMOTER <130> KU1-1P <160> 5 <170> Kopatentin 2.0 <210> 1 <211> 1200 <212> DNA <213> artificail sequence <400> 1 aaaaaagcag agacttggac agtgacttct atttatgaag tatcaatata ttttaattct 60 ttttattatg attttattat tatatgcaat ctggaatatt ttttttgaca tttctcagaa 120 tatcagagat tttagatttt aaaagtgttg ctaaattttt atataacatt tctttattat 180 gtttccctga taaacctaaa gtttgtgata tttaaatgaa aatccaaatt agggtattaa 240 ttggattcat ataaaactta ttttatggca tgttattata tttctcaaat ttgtttagtt 300 tggctaaaca aaaatttgat ataagaaaaa cattaattac attaacctaa aatatcatag 360 ctaaattaag tgggtgatat cattgcaact tttaatacat ttcaccaatt tttatctata 420 tatcatttat ttatttttac tttttttatg taatcatgaa attatgaata tgtgatccat 480 aatctcttta gtcccagaat tagactgata atatttgtgt caagctcacg gttgtagatc 540 atcttataac aaaacttgaa ctcggtttcg gtatccttga atcattgtaa acaacattca 600 tctaattggt taaaccaata caatgagtta ttaaataagt aaatttatcg tcttgttatt 660 atttaaaata tataggtaaa tgtcaacaca ttgaatattg aaaccatttg ataaagtggg 720 ttgtaatttg taaaatacta aaccatatga cagggtttac tatggattta aattgattct 780 atgtgacgtt ctggtgcatg ctcaaccatg agcttatatt agattaaaat cttgttctat 840 cgaaccggtt tgaaactcat cactcaaagc cattttgaat tcagtttggt gaataaactt 900 ataacatatc taagtttcat aaataagtat tgttatatat catcaattta tttatcaaga 960 ttaacatacc tcacacagtt ttttaataaa aaacatgcag catgcagact taaatatggg 1020 ttatgttagt cctcattcca ctctcgacaa gaccacaacc cacaccatca gaattaatat 1080 ttcctcggat acaatactgt ccgaaagtgt agtcacacct caacactagc ttgtgcatca 1140 tttgaaaagt agttgaatcc tacaacagac tagtcaaaac aaactcaccc agcaccatga 1200                                                                         1200 <210> 2 <211> 32 <212> DNA Artificial sequence <400> 2 ggcgaagctt aaaaaagcag agacttggac ag 32 <210> 3 <211> 28 <212> DNA Artificial sequence <400> 3 atttggatcc tcatggtgct gggtgagt 28 <210> 4 <211> 19 <212> DNA Artificial sequence <400> 4 gtaaaccccg tcttcaacg 19 <210> 5 <211> 23 <212> DNA Artificial sequence <400> 5 cgccaatagt accgtaagga taa 23

Claims (6)

1. A promoter comprising a nucleotide sequence represented by SEQ ID NO: 1 and inducing a main pathogen-specific expression of a plant. An expression vector comprising the promoter of claim 1. An expression vector comprising the promoter of claim 1 and a gene operably linked thereto. A transformant comprising any one of the promoter of claim 1, the vector of claim 2, and the vector of claim 3. 2. A plant transformation composition comprising the promoter of claim 1, the vector of claim 2, and the vector of claim 3. A primer set consisting of a forward primer consisting of SEQ ID NO: 2 and a reverse primer consisting of SEQ ID NO: 3 for amplifying a DNA fragment containing the nucleotide sequence shown in SEQ ID NO: 1;

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