KR101726214B1 - Light Inducible Promoter Enhanced Light Inducible Activity and Gene Expression System Comprising The Same - Google Patents
Light Inducible Promoter Enhanced Light Inducible Activity and Gene Expression System Comprising The Same Download PDFInfo
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Abstract
The present invention relates to an improved promoter using a SORLIP element and a gene expression system containing the promoter. When the polynucleotide for promoting optical activity of the present invention is used, the light-oil activity of the light-oil promoter containing the SORLIP element can be greatly enhanced. When the transformant of the present invention is used, The target protein can be effectively expressed while being controlled by irradiation.
Description
The present invention relates to an improved promoter using a SORLIP element and a gene expression system containing the promoter.
Recent attempts to use microalgae in the latest biotechnology, such as biofuels development, carbon dioxide emission reduction, and production of high value added materials, are underway to analyze the nucleotide sequence of genes. As part of this effort, attempts have been made to introduce genes that are useful in existing microalgae to produce the desired traits. However, microalgae have problems in transformation techniques such as low transformation efficiency and low expression efficiency of introduced genes (Lumbreras et al., Plnat J. 1998; 14: 441-447).
As one of important means for solving such problems, various promoters for microalgae have been developed, and microalgae-derived promoters have been used to induce effective transformation and gene expression in the case of using a known promoter for higher plants (Walker et al., J. Applied Phycol. 2005; 17: 363-368). Recent studies and publications have shown that algae of the genus Clamidomonas (Schroda et al., The Plant Journal 2000; 21 (2): 121-131) and dunalella spp. (Li et al., Mol Biol. ; 37: 1143-1154) were also the results of such studies.
The promoter derived from the genus Clamidomonas sp. Disclosed in Schroda et al. Is a promoter that induces gene expression by heat shock mainly by fusion of two different promoters. In the case of using the promoter, there is a problem that if heat is applied to induce the expression of the gene, the growth or metabolic process of the algae may be impaired.
The promoter derived from Dulnellella spp. Disclosed in Li et al. Is a promoter that induces gene expression under conditions of high salt concentration (2M NaCl). The promoter can be applied only to a habitant species, and thus the application range is narrow.
Therefore, it is inevitable to develop a promoter which can be applied to various kinds of living organisms and can control the expression of genes easily, without imposing a burden on the growth or metabolic process of the organism, while broadening the range of application.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
The present inventors have made extensive efforts to develop a mineral oil promoter having enhanced mineral oil activity. As a result, in the light-oil promoter containing the SORLIP element, the present inventors have completed the present invention by confirming that the light oil activity is greatly improved when the SORLIP element portion is replaced with two or more SORLIP element repeat sequences having a specific sequence.
Accordingly, it is an object of the present invention to provide a polynucleotide for promoting the light-oil activity of a light oil promoter comprising a SORLIP element.
It is another object of the present invention to provide a method for promoting mineral oil activity of an optical oil-based promoter comprising a SORLIP element.
It is another object of the present invention to provide a novel mineral oil promoter.
It is still another object of the present invention to provide a gene construct containing a novel mineral oil promoter.
It is still another object of the present invention to provide an expression vector comprising the above-described gene construct.
It is yet another object of the present invention to provide an expression vector comprising a novel light-inducible promoter and a multiple cloning site (MCS).
It is still another object of the present invention to provide a transformant transformed with the above-mentioned expression vector.
It is still another object of the present invention to provide a method for producing an exogenous protein using the above transformant.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a polynucleotide for promoting an optical activity of an optical oil promoter comprising a SORLIP element comprising a nucleotide sequence represented by the following general formula:
[Formula 1]
5'-GCCAC- (X a -GCCAC) n -3 '
Wherein G represents guanine, C represents cytosine, A represents adenine, Xa represents a nucleic acid sequence consisting of 15-25 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine, Wherein n is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the general formula 1 sequence are each the same or different.
The present inventors have made extensive efforts to develop a mineral oil promoter having enhanced mineral oil activity. As a result, in the light-oil promoter containing the SORLIP element, the light oil activity was greatly improved when the SORLIP element portion was replaced with two or more SORLIP element repeat sequences having a specific sequence.
As used herein, the term "promoter " means a DNA sequence capable of regulating the expression of a coding sequence or functional RNA. The promoter region can be easily recognized by those skilled in the art. That is, the start codon of the estimate including the ATG motif is confirmed, and the upstream is the estimated promoter region from this start codon. A promoter consists of contiguous and remote upstream elements. Adjacent elements typically include a TATA box that allows RNA polymerase II to initiate synthesis of RNA at suitable transcription initiation sites. The distant element contains a regulatory sequence, also called an enhancer, which is an additional regulatory element involved in tissue-specific or time-specific expression in the upstream region of the TATA box. An enhancer is a DNA sequence capable of promoting the activity of a promoter and may be a unique element of the promoter or a heterologous element inserted to enhance the tissue-specificity or expression level of the promoter. The promoter may be derived entirely from the original gene, or it may consist of different elements derived from different promoters found in nature, or may even contain synthetic DNA. It will be understood by those skilled in the art that each different promoter may direct expression of the gene in different tissue or cell types, or at different stages of development, or in response to different environmental conditions. A promoter that expresses a gene for most of the time in most cell types is called a "constitutive promoter ". Various types of new promoters useful in plant cells have been continuously discovered; A number of examples of promoters are disclosed in a compilation by Okamuro and Goldberg (1989, Biochemistry of Plants 15: 1-82). It is recognized that, in most cases, the exact border of the regulatory sequence is not completely defined, so that certain mutated DNA fragments have the same promoter activity.
"SORLIP" of the present invention is described in "Hudson, Matthew E., and Peter H. Quail." Identification of promoter motifs involved in phytochrome A-regulated gene expression by combined analysis of genomic sequence and microarray data. " Plant physiology 133.4 (2003): 1605-1616. "As a result of the statistical analysis on the promoter of a gene group whose gene expression is regulated by conventional light, the upstream element, which frequently exists and is expected to play an important role in the mineral oil pathway, to be.
Using the LIP promoter derived from Dannaliella, the present inventors have found that there is a site that plays a major role in the mineral oil mechanism in the sequence at the -100 to -125 nucleotide position, and the GCCAC sequence, the SORLIP element, . The present inventors have further found that when the GCCAC sequence is arbitrarily replaced with a sequence repeated two or more times, the light-oil activity is improved, and that the distance between the repeated sequences has a significant effect on the improvement of the mineral oil activity Respectively.
The "light oil promoter comprising the SORLIP element" of the present invention is a promoter in which the expression of another nucleic acid sequence operatively linked to the promoter by light irradiation is induced, and includes a promoter containing a GCCAC nucleotide sequence known as SORLIP element . Specifically, for example, a known LIP promoter (Park, Seunghye, et al. "Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii." Planta 238.6 (2013): 1147-1156.), PkHMGR promoter Functional & integrative genomics 14.1 (2014): 191-203. & Quot; The GATA and SORLIP motifs in the 3-hydroxy-3-methylglutaryl-CoA reductase promoter of Picrorhiza kurrooa for the control of light- ), PyHLIP promoter (Kong, Fanna, et al., "Cloning and characterization of the HLIP gene encoding high light-inducible protein from Porphyra yezoensis" Journal of applied phycology 24.4 (2012): 685-692), AtELIP1,2 Promoter (Alvarez-Canterbury, Ana M. Rus, et al., "A double SORLIP1 element is required for high light induction of ELIP genes in Arabidopsis thaliana." Plant molecular biology 84.3 (2014): 259-267. But is not limited thereto.
As used herein, the term "operatively linked" refers to a functional linkage between an array of nucleic acid expression control sequences, for example, an array of promoter, signal sequence, or transcription factor binding sites and other nucleic acid sequences, Whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequences.
In one embodiment of the invention, the polynucleotide element of the invention is included in the light-oil promoter by replacing the SORLIP element of the light-oil promoter comprising the SORLIP element. The inventors of the present invention confirmed that the light oil activity was greatly increased when the polynucleotide element represented by the general formula 1 was substituted for the existing SORLIP element. When the polynucleotide element represented by the general formula 1 substitutes the existing SORLIP element, part or all of the sequence existing at the 5'-terminal side of the existing SORLIP element can be removed together.
[Formula 1]
5'-GCCAC- (X a -GCCAC) n -3 '
Wherein G represents guanine, C represents cytosine, A represents adenine, Xa represents a nucleic acid sequence consisting of 15-25 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine, Wherein n is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the general formula 1 sequence are respectively identical or not identical. The present inventors have found that when a plurality of SORLIP elements and GCCAC sequences are spaced by about 15-25 nucleotide sequences, they exhibit the highest effect of enhancing optical activity. Preferably 17-21 nucleotides in length.
The n in the general formula (1) of the present invention is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the general formula 1 sequence are respectively identical or not identical. When n is an integer of 1 to 3, it means that the SORLIP sequence in the promoter can be repeated 2-4 times. The Xa sequence is a sequence for separating a certain distance between SORLIP sequences. It is important that the Xa sequence is composed of 17-21 nucleotides and does not require specific sequence information, and is maintained at a certain distance. In one embodiment, the Xa sequence is a sequence having a sequence from the first nucleotide bound to the 5 'end of the SORLIP element to the 15th to 25th nucleotides in the 5' end direction in the naturally occurring promoter nucleic acid sequence including the SORLIP element But is not necessarily limited thereto.
In one embodiment of the present invention, n in the general formula (1) of the present invention is an integer of 1 to 2. As described above, the inventors of the present invention have confirmed that the light oil activity is increased by repeating a single SORLIP element two or more times apart at a predetermined distance, and it is preferable that the SORLIP element can be repeated 2-3 times .
In one aspect of the present invention, n in the general formula (1) of the present invention is 1. The inventors of the present invention have found that the SORLIP element having two repetitions or the SORLIP element having three repetitions can be preferably used.
In accordance with another aspect of the present invention, the present invention provides a method of modulating the light oil activity of a light oil promoter comprising a SORLIP element, comprising the step of substituting the SORLIP element of the light oil promoter comprising the SORLIP element with the polynucleotide of the invention described above Thereby providing an activity enhancement method. When a polynucleotide of the present invention is substituted with a conventional SORLIP element, part or all of the sequence existing at the 5'-terminal side of the existing SORLIP element can be removed together.
Polynucleotides of the invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Known homologous recombination (HR) and nonhomologous end-joining (NHEJ), Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / cas System, and / or engineered meganuclease re-engineered homing endonucleases ("Esvelt, KM .; Wang, HH. (2013)." Genome " Tan, WS .; Carlson, DF .; Walton, < RTI ID = 0.0 > MW .; Fahrenkrug, SC .; Hackett, PB. (2012), "Precision editing of large animal genomes." Adv Genet 80: 37-97 doi: 10.1016 / B978-0-12-404742-6.00002-8. PMC 3683964. PMID 23084873. and Puchta, H., Fauser, F. (2013). "Gene targeting in plants: 25 years later." Int. J. Dev. Biol 57: 629-637. 10.1387 / ij db.130194hp. ").
Even if all of the nucleic acid sequences bound to the 5 ' end of the original SORLIP element are removed, the effect of the present invention is not largely affected and thus some or all of them can be deleted.
The method of enhancing the light-oil activity of the light oil-repellent promoter comprising the SORLIP element of the present invention corresponds to the method of using the polynucleotide for promoting the light-oil activity of the light oil promoter comprising the SORLIP element, which is another embodiment of the present invention described above, For duplicate content, reference is made to its contents and omitted to avoid undue complexity as described herein.
According to one aspect of the present invention, there is provided a light-oil promoter comprising a nucleotide sequence represented by the following general formula 2:
[Formula 2]
5'-X b -GCCAC- (X a -GCCAC) n -X c -3 '
Wherein G represents guanine, C represents cytosine, A represents adenine, and X a represents a nucleic acid sequence consisting of 15-25 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine , wherein X b represents a nucleotide sequence consisting of from 0 to 15 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine, wherein X c represents a -1 to -101 of the nucleotide sequence LIP promoter, wherein n is Is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the
The "light-oil promoter" of the present invention induces expression of a gene operatively linked to the downstream of the promoter, that is, the 3 'end of the promoter sequence, by irradiation of light, Means a promoter capable of regulating the expression level of a gene.
In the
X b in the
In one embodiment of the invention, it X a of the present invention is a nucleic acid sequence consisting of 17-21 nucleotides. The 17-21 sequences specifically include, for example, nucleotides from the nucleotide at position -107 to the nucleotides at positions 17 to 21 in the 5 'terminal direction, which is the first nucleotide bound to the 5' end of the SORLIP element of the LIP promoter Sequence, but is not necessarily limited thereto.
In one embodiment of the present invention, n in the general formula (2) of the present invention is an integer of 1 to 2. As described above, the inventors of the present invention have confirmed that the light oil activity is increased by repeating a single SORLIP element in a state where the SORLIP element is spaced apart by two or more times, and preferably, the SORLIP element can be repeated 2-3 times Respectively.
In one embodiment of the present invention, n in the general formula (2) of the present invention is 1. As described above, the inventors of the present invention can preferably use the two-time repeated SORLIP element or the three-time repeated SORLIP element, but the present inventors have found that the SORLIP element has the highest light oil promoter activity when the SORLIP element is repeated twice.
In one embodiment of the present invention, the nucleotide sequence represented by the
According to one aspect of the present invention, there is provided a gene construct in which a gene coding for an exogenous protein is operably linked to the above-described optically permissive promoter of the present invention.
The term "exogenous protein" of the present invention means a protein to be produced, and may be any kind of protein whose base sequence is known. In particular, the exogenous protein may be a hormone, a hormone analogue, an enzyme, an enzyme inhibitor, an antibody, or a fragment thereof.
The gene encoding the exogenous protein is operably linked to the light-inducible promoter of the present invention. The operably linked means that expression of the exogenous protein is linked so that it can be regulated by the activity of the photoinduced promoter. Thus, the "genetic construct" in which a gene encoding an exogenous protein is operatively linked to the light inducible promoter is an expression cassette that functions as a unit for expressing the gene encoding the exogenous protein.
The gene construct may be included in an expression vector to control the expression of the gene encoding the foreign protein light-dependent.
According to one aspect of the present invention, there is provided an expression vector comprising the above-described gene construct of the present invention.
As used herein, the term "expression vector" means a vector capable of expressing a foreign protein of interest in a host cell, the vector comprising an essential regulatory element operably linked to the expression of the gene insert. Suitable expression vectors include signal sequence or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoter, operator, initiation codon, termination codon, polyadenylation signal and enhancer, and may be prepared in various ways depending on the purpose.
The expression vector of the present invention may further comprise a selection marker for selecting a host cell containing the vector. The selectable marker may be any known type of selectable marker, and may include, but is not limited to, an antibiotic resistance gene or a light emitting gene. Specifically, for example, the expression vector of the present invention may further carry a gene encoding a reporter molecule (e.g., luciferase and? -Glucuronidase). In addition, the expression vector of the present invention can be used as a selection marker for a gene resistant to antibiotics (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) (for example, neomycin phosphotransferase (nptII) Gomycin phosphotransferase (hpt), etc.).
The expression vector of the present invention may be one which introduces the light tolerant promoter of the present invention into all conventional expression vectors, and may be a recombinant expression vector artificially designed by a known method. The production of an expression vector by introducing the above-described light-oil promoter can be easily carried out according to a known method by those skilled in the art.
The vector for producing the expression vector may be any plasmid vector, cosmid vector, bacteriophage vector, and viral vector, as long as it is capable of introducing the light tolerant promoter of the present invention. In particular, it is preferable to use a vector stably present in the host cell such as microalgae or higher plants and having a large number of copies.
The expression vector of the present invention may be introduced into a host cell to transform the host cell.
Such transformation can be easily carried out by a person skilled in the art by conventionally known transformation techniques, and the transformation technique can be carried out by a transformation method using glass beads known by Kindle (1990), a protoplast using a calcium / polyethylene glycol method Particle bombardment, electrophoresis, agrobacterium-mediated method, gene gun or physical introduction method, and the like. The transformation technology may be appropriately selected by those skilled in the art according to the type and characteristics of the host cell.
The host cell for transformation with the above expression vector is preferably a microalgae or a higher plant, more preferably Clamidomonas or Dernalia, but is not limited thereto. The RNA polymerase capable of recognizing the promoter of the present invention Are all available. The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).
According to one aspect of the present invention, there is provided a transformant transformed with the above-described expression vector of the present invention. The transformant of the present invention is preferably a transformant obtained by transforming a microalgae or a higher plant as a host and transforming with the above-described expression vector as described in the detailed description of the expression vector of another embodiment of the present invention.
According to one aspect of the present invention, the present invention provides a method for producing an exogenous protein comprising the steps of:
(a) culturing the transformant described in another embodiment of the present invention; And
(b) irradiating the cultured transformant with light.
The transformant of the present invention is a transformant transformed with an expression vector comprising the light-inducible promoter described in another embodiment of the present invention, which is cultured to amplify the number of transformants, To induce expression of a nucleic acid sequence operably linked to a light-oil promoter. The nucleic acid sequence operably linked to the light-oil promoter may be a nucleic acid sequence encoding an exogenous protein for mass production.
The culture of step (a) may be suitably performed by a person skilled in the art depending on the type and characteristics of the transformant, the culture method, the culture medium and the culture conditions.
The light of step (b) can be appropriately selected by a person skilled in the art depending on the target production amount of the foreign protein, and the light intensity may be 10 to 1000 μmol photon /
In accordance with another aspect of the present invention, the present invention provides a mutagenic promoter as described in another embodiment of the present invention, and (b) a multiple cloning site in which a gene encoding an exogenous protein can be operably inserted into the promoter sate, MCS). Using the multiple cloning site, a gene encoding an exogenous protein can be easily inserted into the expression vector. The "exogenous protein" of the present invention is as described above.
According to one aspect of the present invention, there is provided a transformant transformed with an expression vector comprising the above-mentioned multiple cloning site. The transformants and transformants of the present invention are the same as those described for the transformation of host cells using the expression constructs of the present invention and the expression vectors containing the gene constructs of the present invention and the transformation products thereof.
According to another aspect of the present invention, the present invention provides a method for producing an exogenous protein comprising the steps of:
(a) culturing the transformant transformed with an expression vector comprising MCS of the present invention; And
(b) irradiating the cultured transformant with light.
The method for producing an exogenous protein of the present invention is as described in the method for producing a protein using a transformant transformed with an expression vector comprising a gene construct, which is another embodiment of the present invention described above, Omission is avoided in order to avoid the excessive complexity of the present specification.
The features and advantages of the present invention are summarized as follows:
(a) The present invention provides polynucleotides for promoting the light-oil activity of an optical oil-repellent promoter comprising a SORLIP element.
(b) The present invention provides a method for promoting mineral oil activity of an optical oil-based promoter comprising a SORLIP element.
(c) The present invention provides a novel mineral oil promoter.
(d) The present invention provides a gene construct comprising a novel light oil promoter.
(e) The present invention provides an expression vector comprising the aforementioned gene construct.
(f) The present invention provides an expression vector comprising a novel light-inducible promoter and a multiple cloning site (MCS).
(g) The present invention provides a transformant transformed with the aforementioned expression vector.
(h) The present invention provides a method for producing an exogenous protein using the above-mentioned transformant.
(i) When the polynucleotides for promoting the activity of the light oil activity of the present invention are used, the light oil activity of the light oil promoter containing the SORLIP element can be greatly enhanced.
(j) When the transformant of the present invention is used, the target protein can be effectively expressed while controlling the expression amount of the target protein by light irradiation.
FIG. 1 shows experimental results of measuring the luciferase of each promoter according to the light intensity in order to find the light-oil element of the LIP which is the light-oil promoter.
FIG. 2 shows the result of analyzing the promoter sequence between 100p and 200p by PLACE signal scan search.
Figure 3 shows the results of truncation analysis between 200p and 100p of the LIP promoter.
FIG. 4 shows the result of measuring the optical transparency of a new promoter made by repeating two or three SORLIP element regions.
FIG. 5 shows the results of measurement of the liver mineralogical property of a new promoter made by changing the SORLIP elements of the SORLIPX2 promoter at intervals of 0, 7, 17, 24, and 34 nucleotides, respectively.
FIG. 6 shows experimental results comparing and measuring the intensity of SORLIPX2, LIP-400P, and AR promoter according to the intensity of light.
Figure 7 shows the sequence of the SORLIPX2 promoter.
Figure 8 shows a map of a vector utilizing the SORLIPX2 promoter.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example
Example 1: Discovery of a SORLIP motif that plays a crucial role in the mineral oil pathway of the light-oil promoter
The mineral oil promoter LIP (LIP) derived from the marine microalga DINALELLA (Kim, Minjung, et al., "Gene expression profiling of Dunaliella sp. Acclimated to different salinities." Phycological research 58.1 By reducing the length of the promoter, an attempt was made to find out which part plays an important role in the mineralogical mechanism (see FIG. 1). Experiments were conducted to measure the activity of the promoter by observing its activity by attaching luciferase to the promoter. A novel promoter of 300p, 200p, and 100p was prepared by truncation analysis in which a promoter having a length of about 400 bp was confirmed to have a size of 100p by using a preliminary study and a luciferase was ligated for two hours . It was found that the optical mineralogical mechanism disappeared between 100p and 200p, and it was confirmed that a part that plays a crucial role in the mineral oil mechanism of the LIP promoter exists between 100p and 200p. This can be found on the PLACE (A Database of Plant Cis-acting Regulatory DNA Elements) website (https://sogo.dna.affrc.go.jp/cgi-bin/sogo.cgi?lang=en&pj=640&action=page&page=newplace) As a result of a signal scan search, it was confirmed that there is a SORLIP sequence as a photoreactive element related to phytochrome (see FIG. 2).
Example 2: Confirmation of the light oil mechanism of the ensured SORLIP element
In order to confirm that the ensured SORLIP element plays a crucial role in the mineral oil mechanism in the LIP promoter, truncation analysis was performed in the same manner at 25 bp intervals in the promoter between 200p and 100p. As a result, it was confirmed that the SORLIP element existing between 125p and 100p is an element causing light mineralization in the LIP promoter (see FIG. 3).
Example 3: Fabrication of novel mineral oil promoter with improved function using SORLIP element
A new promoter was constructed using the ensured SORLIP element. The SORLIP element region which was present in one of the known LIP promoters (Park, Seunghye, et al., Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii. Planta 238.6 (2013): 1147-1156. Was repeated two times or three times at intervals of 19 nucleotides. As a result, it was confirmed that the promoter repeated two times with SORLIP showed the most increase in the optical coherence activity (see FIG. 4). In order to investigate the effect of the spacing between the two SORLIP element regions on the expression of the promoter based on the SORLIPX2 promoter (SORLIPX2), the distance between the two SORLIP elements was 2, 9, 19, 26, 36 As a result of measuring the activity of luciferase by the same experimental method, the SORLIPX2 promoter (19 gap SORLIPX2 promoter) at intervals of 19 nucleotides showed the strongest activity (see FIG. 5). It was confirmed from this experiment that the SORLIP element plays a crucial role in the mineralogical mechanism and that the SORLIPX2 promoter, which is repeated twice at 19 nucleotide intervals, has a much greater increase in the optical activity than the conventional promoter .
Example 4: Comparison between a novel SORLIP promoter and a conventional promoter
In order to confirm the increase of the enhanced optical activity of the SORLIPX2 promoter and to observe its intensity, comparative experiment with the conventional vectors was performed. As the comparative group, the AR promoter, which is one of the promoters known to have the strongest activity among the
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
<110> IUCF-HYU (Industry-University Cooperation Foundation Hanyang University) <120> Light Inducible Promoter Enhanced Light Inducible Activity and Gene Expression System Comprising The Same <130> PN150368 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> DNA <213> SORLIP sequence from LIP promoter <400> 1 gccac 5 <210> 2 <211> 139 <212> DNA <213> Artificial Sequence <220> <223> Artificial Light Inducible Promoter Enhanced Light Inducible Activity <400> 2 gggctccggg ccacacattt gcccgggctc cgggccacac atttgccagc ctgaaaactt 60 gccaaaaacc actcatcatc aaaacaacaa aagcttcaat caaaactcgt tcctacaccc 120 acacgaaccg acccgaaca 139 <210> 3 <211> 439 <212> DNA <213> LIP promoter from Dunaliella <400> 3 cccagtgtag gtgtctttgc tagtgtactc ctacacgtcc tcaatccacg agcgtgcaca 60 ctaaacactt gtgcatgcac tgtcaccagg ctttggcagc gccaggaggt tcttaaagtg 120 acatccgtgt cccgcagcaa cttacattga ccaacagtca acaccctcca accctcacag 180 gttcaagaca catacaacac tgttcactca ctcgtgattt gcaaaatgta aagctttggc 240 cctcttggct ttttttttct cacgggcagc tcacccaccg actcactcac gcactcacca 300 acagagcgtg gcctcggagc gtgagggctc cgggccacac atttgccagc ctgaaaactt 360 gccaaaaacc actcatcatc aaaacaacaa aagcttcaat caaaactcgt tcctacaccc 420 acacgaaccg acccgaaca 439 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> 2 gap SORLIPx2 sequence <400> 4 gggctccggg ccacgggcca cacatttgcc a 31 <210> 5 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> 9 gap SORLIPx2 sequence <400> 5 gggctccggg ccacgggctc cgggccacac atttgcca 38 <210> 6 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> 19 gap SORLIPx2 sequence <400> 6 gggctccggg ccacacattt gcccgggctc cgggccacac atttgcca 48 <210> 7 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 26 gap SORLIPx2 sequence <400> 7 gggctccggg ccacgggctc cacatttgcc cgggctccgg gccacacatt tgcca 55 <210> 8 <211> 139 <212> DNA <213> Artificial Sequence <220> <223> 36 gap SORLIPx2 sequence <400> 8 gggctccggg ccacacattt gcccgggctc cgggccacac atttgccagc ctgaaaactt 60 gccaaaaacc actcatcatc aaaacaacaa aagcttcaat caaaactcgt tcctacaccc 120 acacgaaccg acccgaaca 139
Claims (18)
[Formula 1]
5'-GCCAC- (X a -GCCAC) n -3 '
Wherein G represents guanine, C represents cytosine, A represents adenine, Xa represents a nucleic acid sequence consisting of 15-25 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine, Wherein n is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the general formula 1 sequence are respectively identical or not identical.
[Formula 2]
5'-X b -GCCAC- (X a -GCCAC) n -X c -3 '
Wherein G represents guanine, C represents cytosine, A represents adenine, and X a represents a nucleic acid sequence consisting of 15-25 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine , Wherein X b represents a nucleic acid sequence consisting of 0-15 nucleotides selected from the group consisting of adenine, guanine, cytosine and thymine, and X c represents a nucleic acid sequence consisting of -1 to -10 of the LIP promoter of SEQ ID NO: 101 nucleotide sequence, wherein n is an integer of 1 to 3, and when n is 2 or more, a plurality of Xa sequences contained in the general formula 2 sequence are identical or not identical to each other.
(a) culturing the transformant of claim 14; And
(b) irradiating the cultured transformant with light.
(a) culturing the transformant of claim 17; And
(b) irradiating the cultured transformant with light.
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