WO2013180488A1 - Light-inducible promoter and gene expression system containing same - Google Patents

Description

 【Specification】

 [Name of invention]

 Photo-inducible promoter and gene expression system comprising the same

 The present invention was made by the task number 2011-0031999 under the support of the Ministry of Education, Science and Technology, and the research management specialized agency of the task is the Korea CO2 Collection and Treatment Research and Development Center,

"Global Science R & D Project (Korea CCS 2020 Project)", Project Name "Development of High-Efficiency Carbon Dioxide Fixed Microalgae through Molecular Biology Improvement," Organized by Hanyang University Industry-University Cooperation Group, Research Period 2011. 11. 01-2012. 05. 31.

 This patent application is filed with the Korean Patent Application No. 10-2012-0059411 filed with the Korean Patent Office on June 1, 2012 and the Korean Patent Application No. 10-2012-0149832 filed with the Korean Patent Office on December 20, 2012. Priority is claimed, the disclosures of which are incorporated herein by reference.

 The present invention relates to a novel promoter, and more particularly to a light inducible promoter and a gene expression system comprising the same.

Background Art

 In recent years, efforts have been made to analyze the nucleotide sequence of genes with the attempt to use microalgae in the latest biotechnology such as biofuel development, carbon dioxide reduction, and high value-added material production. As part of this effort, attempts have been made to introduce genes useful in existing microalgae to exhibit 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 the important means to solve this problem, various promoters for microalgae are being developed, and promoters derived from microalgae By using this method, it was possible to induce more efficient transformation and gene expression than using a conventionally known promoter for higher plants (Walker et al., J. Applied Phycol. 2005; 17: 363-368). Recently studied and published Chlamydomonas algae (Schroda et al., The Plant Journal 2000; 21 (2): 121-131) and Dunalella algae (Li et al., Mol Biol. Rep. 2010 37: 1143-1154), a study of promoters is one result of this study.

 Promoter derived from Chlamydomonas algae published in Schroda et al. Is a fusion of two different promoters and is a promoter that mainly induces gene expression by thermal stratification. In the case of using the promoter, if heat is added to induce the expression of a gene, there is a problem that can give rise to the growth or metabolism of the bird.

 Promoter derived from Dunalella genus alga published in Li et al. Is a promoter that induces the expression of genes under high salt conditions (2M NaCl). The promoter is only applicable to basophilic species, there is a narrow range of applications.

 Accordingly, the development of a promoter that can be easily applied to various kinds of living organisms and can regulate gene expression without burdening the growth or metabolic process of the living organisms is necessary. Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained. [Content of invention]

 Problem to be solved

It is an object of the present invention to provide a gene and a gene expression system including the same, which can be applied to various kinds of organisms, and which can easily regulate expression of a gene by irradiation of light. Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

[Measure solution]

 In order to achieve the above object, one aspect of the present invention is SEQ ID NO:

Provided is a light inducible promoter comprising the nucleotide sequence described as 12.

 In addition, another aspect of the present invention to achieve the above object provides an expression vector comprising the photo-inducible promoter, and a transformant transformed with the expression vector.

 In addition, another aspect of the present invention to achieve the above object is an expression construct, the expression construct operably linked to a gene encoding a foreign protein to a light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12, An expression vector including a truck, and a transformant transformed with the expression vector are provided.

 In addition, another aspect of the present invention to achieve the above object is the step of culturing the transformant; And it provides a method for producing a foreign protein comprising the step of irradiating the cultured transformant with light. 【Effects of the Invention】

 Since the light inducible promoter of the present invention induces gene expression by irradiation of light and regulates the expression level of the gene according to the light intensity, it is possible to easily control the expression of the gene without burdening the growth or metabolic process of the organism. There is a wide range of advantages that can be applied to various kinds of organisms.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description. [Brief Description of Drawings]

La and lb are nucleotide sequence analysis results of a _P GEM ^(R) -T easy vector cloned with LSIP_P in Example <1-2> of the present invention.

 The shaded portion of gray represents the first axon;

The nucleotide sequence of the remainder except for the shaded portion of gray is the nucleotide sequence of！ _^ ！？.

bar clawing cbr 프로모터의 뉴클레오타이드 서열을 비교한 그림으로서 , 2 is Dunaliella Esp. LSIP_P and Dunaliella Badhama at / 2a // e // a sp.

Figure comparing the nucleotide sequences of the bar clawing cbr promoter,

 bar represents the nucleotide sequence of Dunalella haevil (Zte // e // a bardawil) ^ cbr promoter;

 sp shows the nucleotide sequence of！ ^^？ of Dunaliella sp.

 3 is a vector map of pN7-AR_P-LUC—TPsaD, which is an expression vector for chlamidomonas,

 the nic7 gene represents the quinolinate synthetase gene which acts as a selection marker upon transformation;

 AR-P represents a recombinant promoter from Chlamydomonas; Renilla lucif erase CDS represents a gene encoding an enzyme that causes light to be degraded while cleaving a substrate (Coelenterazine);

 PsaD-T represents the terminator portion of the PsaD gene from Chlamydomonas.

 4 is a vector map of an expression vector in which 1LSIP_P, 4LSIP_P, 8LSIP_P, 17LSIP_P, or Bar-CBR_P is inserted into a region from which AR_P is removed in pN7-AR- P-LUC-TPsaD of FIG.

Figure 5 Isolates from transformants introduced pN7-lLSIP_P-LUOTPsaD, _P N7-4LSIP_P-LUC-TPsaD, pN7-8LSIP_P-LUC-TPsaD, _P N7-17LSIP_P-LUC-TPsaD and pN7-Bar_CBR_P—LUC-TPsaD As a gel photograph confirming whether the vectors were normally introduced by performing PCR on genomic DNA and confirming that respective bands corresponding to the 936 bp luciferase gene were detected. + And-mean the plasmid vector used for transformation and the original Chlamydomonas without transformation;

 1 to 6 are 1LSIP_P, 7 to 12 are 4LSIP_P, 13 to 18 are 8LSIP_P, 19 to 24 are 17LSIP_P, and 25 to 30 are Bar-CBR ᅳ P, respectively.

 6 shows 1LSIP_P. It is a graph measuring the expression level according to the light intensity of luciferase which is controlled by 4LSIP_P, 8LSIP_P, 17LSIP_P and Bar-CBR_P.

 7 is 1LSIP_P. Luminescence ratio is a graph measuring the expression ratio according to the irradiated light intensity of luciferase under the control of 4LSIP_P, 8LSIP ᅳ P, 17LSIP_P and Bar-CBR_P. Luminescence Amount) I (luminescence amount in cancer treated cells). [Specific contents to carry out invention]

 First, the terms used in the specification of the present invention will be described.

 In the present invention, referred to as' dunaliella ^^ \. DimalieUa sp.) Is renamed from the existing Kim et al. CPhycological Research 2010; 58: 17-28), and the typical Dunaliella salina ¾ / 7M // e // a salina) is newly renamed Meaning a bird in the genus of two nalellia, it is a species of the same species as the conventional dunaliella salina (Z½2a / / e / / a sa / /).

In the present invention, the term 'LSIP Lignt and Salt Inducible Protein)' is a protein having an amino acid sequence of SEQ ID NO. 3 derived from DunalieHa sp., And described by Kim et al. (Phyco logical Research 2010; 58: 17-28). It is known in the literature as the cbr protein (carotenoid biosynthesis related protein). Kim et al. In the literature, the protein having the amino acid sequence of SEQ ID NO: 3 is Dunalella salina (Z¾ / y // e // a salina), algal in another Dunaliella genus and Dunalella haevil 0 -3 // e // 3 bardawil), such as cbr protein and shows a high homology of about 80%, it is named cbr protein. However, unlike the D. salina and D. bardawil species, the Dunaliella sp. ¾7 / // e // a sp.) Species that do not accumulate carotenoids, especially Dunaliella sp. Dunaliala sp. In terms of the function performed by the protein of SEQ ID NO: 3 in ½2a // e // a sp.) Or the association between the protein of SEQ ID NO: 3 and the carotenoid. The protein of SEQ ID NO: 3 derived from ¾ // 7a // e // a sp,) needs to be distinguished from the cbr proteins of D. salina and D. bardawi 1. Therefore, in the present invention, the protein of SEQ ID NO: 3 is renamed as Lignt and Salt Inducible Protein (LSIP), and it is defined as distinguished from the cbr proteins of D. salina and D. bardawi 1.

 As used herein, the term "promoter" refers to a DNA sequence that regulates the expression of a gene encoding a foreign protein operably linked in a particular host cell.

 The expression vector, which is referred to in the present invention, is a vector capable of expressing a foreign protein of interest in a host cell, and means a vector including essential regulatory elements operably linked to express a gene insert. Suitable expression vectors include signal sequences or leader sequences for membrane targeting or secretion in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation Sig ¾ and enhancers and can be prepared in various ways depending on the purpose. .

 The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). Hereinafter, the present invention will be described in detail.

Dunaliella Esp. Originated promoter

 One aspect of the invention is Dunaliella sp. Comprising a nucleotide sequence set forth in SEQ ID NO: 18. Provide a derived promoter.

The Dunaliella sp of the present invention. The derived promoter comprises a nucleotide sequence set forth in SEQ ID NO: 18. The promoter comprises a nucleotide sequence set forth in SEQ ID NO: 18 of 100 bp in length. The nucleotide sequence set forth in SEQ ID NO: 18 induces and enhances the 'expression' of the gene downstream of that, ie, the gene encoding the foreign protein operably linked to the 3 'end of the nucleotide sequence. However, the nucleotide sequence described in SEQ ID NO: 18 is only a minimum unit of a promoter having the property of inducing and enhancing the expression of such genes, and the sequence of the promoter is not limited to the nucleotide sequence of SEQ ID NO: 18. Thus, the promoter may be a nucleotide sequence of 100 bp to 2084 bp in length selected from the nucleotide sequence set forth in SEQ ID NO: 4, to include the nucleotide sequence of SEQ ID NO: 18. As an example, the promoter may be a nucleotide sequence described by SEQ ID NO: 12 having a length of 439 bp, a nucleotide sequence described by SEQ ID NO: 1 having a length of 845 bp, or a nucleotide sequence described by SEQ ID NO: 2 having a length of 1702 bp. It may include.

 The promoter is derived from Dunaliella sp. // e / / a sp.), Preferably present upstream of the gene encoding the LSIP protein of Dunaliella sp., But is not limited thereto. . Thus, all naturally occurring or artificially synthesized nucleotide sequences can be used as the promoter.

In a specific embodiment of the present invention, by operably connecting the luciferase gene (lucif erase) gene to 1LSIP ᅳ P which is a promoter comprising the nucleotide sequence of SEQ ID NO: 18, the expression vector _P N7-lLSIP_P having a map as shown in FIG. -LUC-TPsaD was prepared, and the transformant was prepared by introducing the expression vector into Chlamymonas reinhardtii JL173 cells, which is a heterogeneous heterodyne with Dunaliella sp., Derived from 1LSIPJ ³ . . Thereafter, the transformant is treated with cancer or irradiated with a light of luminous intensity 30, 300 or 600 μπιοΐ photon / m ² / s, respectively, and whether the expression of the reporter gene luciferase and the luminescence ratio ( luminescence ratio) was measured. As a result, the reporter gene luciferase was irradiated with light under 1SLIPSL P of the present invention and Regardless of the light intensity, light was expressed only in the irradiated transformants (see Table 3 and FIG. 6).

 From the above results, the nucleotide sequence described in SEQ ID NO: 18 of the present invention is a basic unit having promoter activity and is stable and highly efficient for a foreign gene operably linked downstream of the nucleotide sequence described in SEQ ID NO: 18 above. It can be seen that can be expressed as.

2. Photo Inductive Promoter

 Another aspect of the invention provides a light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12.

 In addition, another aspect of the present invention provides an expression vector comprising the light inducible promoter, and a transformant transformed with the expression vector.

 The light inducible promoter of the present invention comprises a nucleotide sequence set forth in SEQ ID NO: 12.

 The promoter comprises a nucleotide sequence set forth in SEQ ID NO: 12 of 439 bp in length. The nucleotide sequence of SEQ ID NO: 12 is based on the nucleotide sequence of SEQ ID NO: 18 having a promoter activity, the promoter activity represented by the nucleotide sequence of SEQ ID NO: 18 in the 5 'portion of the nucleotide sequence described in SEQ ID NO: 18 An additional 339 bp long nucleotide sequence that can be adjusted according to the intensity of light to be irradiated is included.

The nucleotide sequence set forth in SEQ ID NO: 12 induces expression of a gene encoding a foreign protein operatively linked downstream thereof, ie, to the 3 'end of the nucleotide sequence by irradiation of light, in particular at the intensity of the light to be irradiated. Accordingly, the expression level of the gene encoding the foreign protein is adjusted. However, the nucleotide sequence represented by SEQ ID NO: 12 is only a minimum unit of a promoter having a property of controlling the degree of gene expression linked downstream thereof according to the light intensity as described above, and the sequence of the promoter It is not limited to the nucleotide sequence of SEQ ID NO: 12. Thus, the promoter is set forth in SEQ ID NO: 4 In the nucleotide sequence, it may be a nucleotide sequence of 439 bp to 2084 bp in length selected to include the nucleotide sequence of SEQ ID NO: 12. As an example, the promoter may be composed of a nucleotide sequence described by SEQ ID NO: 1 having a length of 845 bp or a nucleotide sequence described by SEQ ID NO: 2 having a length of 1702 bp.

 The promoter is Dunaliella sp. 723 /// a sp.), But is preferably present upstream of the gene encoding the LSIP protein of Dunaliella sp., But is not limited thereto. Thus, all naturally occurring or artificially synthesized nucleotide sequences can be used as the promoter.

 The photoinducible promoter may be included in an expression vector to optically regulate expression of a gene encoding a foreign protein operably linked downstream of the promoter.

 The expression vector may further include a multiple cloning site (MCS) through which a gene encoding a foreign protein can be operably inserted into the promoter. By using the multicloning region, a gene encoding a foreign protein can be easily inserted into the expression vector. The expression vector may further include a selection marker for selecting a host cell containing the vector. The selection marker may be any type of selection marker known in the art, and antibiotic resistance genes, light emitting genes, and quinolinate synthetase genes may be used, but is not limited thereto.

 The expression vector may be the introduction of the light inducible promoter in all conventional expression vectors, it may be a recombinant expression vector artificially designed by a known method. The expression vector may be prepared by introducing the light inducible promoter according to a method well known to those skilled in the art.

The vector for the production of the expression vector may be used as long as it can introduce the light inducible promoter, and may be a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector. In particular, it is desirable to use vectors that are stable in host cells, such as microalgae or higher plants, and that have a high copy number. The expression vector may be introduced into a host cell to transform the host cell into the expression vector.

 The transformation can be easily performed by those skilled in the art by a conventionally known transformation technique, the transformation technique is a protoplast using a transformation method using glass beads, calcium / polyethylene glycol method known by Kindle (1990) Transfection, electroporation, microinjection, particle bombardment, electrophration, Agrobacterium-mediated methods, gene guns or physical introduction methods. The transformation technique may be performed by those skilled in the art as appropriately selected according to the type and characteristics of the host cell.

 The host cell for transforming the expression vector is preferably a microalgae or a higher plant, and more preferably, chlamidomonas or dunnaliline, but not limited thereto, and may recognize the promoter of the present invention.

All cells with RNA polymerase can be used.

In a specific embodiment of the present invention, 4LSIP_P, a promoter comprising the nucleotide sequence of SEQ ID NO: 12, 8LSIP_P, a promoter comprising the nucleotide sequence of SEQ ID NO: 1, and 17LSIP_P, a promoter comprising the nucleotide sequence of SEQ ID NO: 2, respectively luciferase (l _uc if _erase) the expression vector _{pN7-4LSIP_P-LUC-TPsaD, P N7-8LSIP_P} -LUC-TPsaD and pN7-17LSIP_P-LUC-TPsaD having a map as to possibly connect the gene activated, and Figure 4 The three expression vectors, pN7-4LSIP_P-LUOTPsaD, pN7-8LSIP_P-LUC-TPsaD and pN7, were prepared and cloned from the two species Klimidomonas Reinhardty Al) human cells, which are heterogeneous with Dunaliella sp. Transformants were prepared by introducing -17LSIP_P-LUC-TPsaD, respectively. Then, the transformant was treated with cancer or irradiated with light of 30, 300 or 600 μιτιοΐ photon / m ² / s of light, respectively, to determine whether the reporter gene luciferase was expressed and the luminescence ratio. . As a result, the reporter gene luciferase was expressed only in the light irradiated transformant, and as the light intensity of the irradiated light was increased, it was confirmed that high luminescence ratio was shown (see FIG. 6 and Table 2). From the above results, the nucleotide sequence set forth in SEQ ID NO: 12, and the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 comprising the nucleotide sequence described in SEQ ID NO: 12 show activity of the light inducible promoter. Able to know. That is, the nucleotide sequence described in SEQ ID NO: 12 serves as the minimum unit of the light inducible promoter activity as described above, and in the nucleotide sequence described in SEQ ID NO: 4, the nucleotide sequence of SEQ ID NO: 12 is selected to be included It can be seen that nucleotide sequences of various lengths also have the activity of such light inducible promoters. In addition, the LSIP_P has been found to be able to optically control the expression of genes encoding foreign proteins in heterogeneous organisms other than the birds of the genus Denaliella from which the LSIP_P is derived, and are widely applied to various species of organisms. It can be seen that.

3. Production of Foreign Proteins Using Photoinducible Promoter

 Another aspect of the invention is an expression construct comprising an expression construct operably linked to a nucleotide sequence encoding a foreign protein to a light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12, an expression vector comprising said expression construct , And transformants transformed with the expression vector.

 In addition, another aspect of the present invention comprises the steps of culturing the transformant; And it provides a method for producing a foreign protein comprising the step of irradiating the cultured transformant with light.

 The expression construct of the present invention comprises a light inducible promoter comprising a nucleotide sequence set forth in SEQ ID NO: 1 and a gene encoding a foreign protein operably linked to the light inducible promoter.

The light inducible promoter is the same as described in the item ". Light inducible promoter." Thus, the descriptions of the light inducible promoters and the item will be omitted. In the following, only the configuration specific to the expression construct will be described. The foreign protein refers to a protein to be produced, and may be any kind of protein in which the nucleotide sequence of the gene is known. In particular, the foreign protein may be a hormone, a hormonal analog, an enzyme, an enzyme inhibitor, a signaling protein or a part thereof, an antibody or a part thereof, a short chain antibody, affibody, a peptide aptamer, a binding protein or a binding domain thereof, an antigen, an adhesion protein, Structural proteins, regulatory proteins, toxin proteins, cytokines, transcriptional regulators, and blood coagulation factors.

 The gene encoding the foreign protein is operably linked to the light inducible promoter. The operably linked means that the expression of the foreign protein is linked such that it can be regulated by the activity of the photoinducible promoter. Thus, the expression construct formed by operably linking a gene encoding a foreign protein to the light inducible promoter becomes an expression cassette functioning as a unit for expressing the gene encoding the foreign protein.

 According to one embodiment of the invention, the expression construct of the invention further comprises a terminator sequence downstream of the nucleotide sequence encoding the foreign protein. Terminator sequences (ie, poly A signal sequences) that may be used in the present invention include various terminator sequences operable in algae known in the art, for example PsaD-T (PsaD from Chlamydomonas). Terminator of the gene).

 The expression construct may be included in an expression vector to optically control the expression of the gene encoding the foreign protein.

 According to one embodiment of the invention, the transformant comprising the expression construct is algae, specifically green algae, for example green algae such as Dunaliella, Chlamydomonas and the like.

 In addition, the expression vector including the expression construct may be introduced into a host cell, and the host cell may be transformed into the expression vector. The transformant formed as described above can be used for the production of the foreign protein.

The foreign protein production method of the present invention comprises the steps of 1) culturing the transformant; And 2) irradiating the cultured transformant with light. The culturing of step 1) may be appropriately performed by those skilled in the art by varying a culture method, a culture medium, and culture conditions according to the type and characteristics of the transformant.

 The light irradiation of step 2) induces the expression of a gene encoding a foreign protein in the transformant cultured in step 1), wherein the gene is regulated by the activity of a light inducible promoter.

 The light of step 2) may be appropriately selected by those skilled in the art according to a target production amount of a foreign protein, and the light intensity of the irradiated light may be 10 to 1000 umol photon / mVs, but is not limited thereto. In addition, the light of step 2) may be appropriately selected by the person skilled in the art according to the target production amount of the foreign protein, the irradiation time of the light may be 1 to 5 hours, but is not limited thereto.

 The foreign protein production method may further comprise the step of separating the foreign protein expressed from the transformant 3).

 By using the expression construct as described above, an expression vector including the same, and a transformant including the expression vector, foreign proteins can be easily produced. Hereinafter, the present invention will be described in detail by way of examples.

 However, the following examples are only for the understanding of the present invention, and the present invention is not limited to the following examples.

Example 1

Cloning and Nucleotide Sequence Analysis of the Light Inducible Promoter LSIP ᅳ P Referring to the literature, Park et al. (Marine Biotechnology, 2006; 8: 120-128) and Kim et al. CPhycological Research 2010; 58: 17-28). Dunaliella Esp. The nucleotide sequence was cloned by cloning the upstream portion of LSIP, a protein known to vary in expression according to the intensity of light at (/ ½3 // e // a sp.), And was named LSIP_P. <1-1> Cloning of light inducible promoter！ ^^ —？

sp.)의 genomic DNA 에서 , GenomeWalker™ Universal Kit (Cat. #638904) (CI ontech, USA)를 이용하여 제조사에서 제공하는 프로토콜에 따라 광 유도성 프로모터인 LSIP_P 를 pGEM^(R)-T easy 백터 (Promega, USA)에 클로닝하여 pGEM^(R)-T-LSIP_P 를 제조하였다. 상기 Kit를 이용하여 상기 ！^ ᅳ？의 클로닝 과정에서， 서열번호 6으로 기재되는 뉴클레오타이드 서열 및 서열번호 7 로 기재되는 뉴클레오타이드 서열을 각각 GSPKgene specific primer 1) 및 GSP2(gene specific primer 2)로 이용하였다. Park et al. (Marine Biotechnology, 2006; 8: 120-128) and Kim et al. CPhycological Research 2010; 58: 17-28.

In genomic DNA of sp.), the light-inducible promoter LSIP_P was converted to pGEM ^(R) -T easy vector (GenoWalker ™ Universal Kit (Cat. # 638904) (CI ontech, USA) according to the manufacturer's protocol. Promega, USA) to prepare pGEM ^(R) -T-LSIP_P. In the cloning process using the kit, the nucleotide sequence shown in SEQ ID NO: 6 and the nucleotide sequence shown in SEQ ID NO: 7 were used as GSPKgene specific primer 1) and GSP2 (gene specific primer 2), respectively. .

 <1-2> Nucleotide Sequence Analysis of the Photoinductive Promoter LSIPJ

 The nucleotide sequence was analyzed by applying LSIP_P cloned in Example <1-1> to Macrogen (Korea). Nucleotide sequence analysis includes GSP1 of SEQ ID NO: 6 and GSP2 of SEQ ID NO: 7, and the Genome Walker ™

Adapter primers provided in the Universal Kit were used.

As a result, it was confirmed that the nucleotide sequence described in SEQ ID NO: 5 was cloned into the pGEM ^(R) -T easy vector (FIGS. La and lb). From the above nucleotide sequence analysis, the sequence present upstream of the first axon sequence of LSIP in the nucleotide sequence described in SEQ ID NO: 5 becomes the nucleotide sequence of LSIP_P, and finally, LSIP_P is described in SEQ ID NO: 4 Was identified as having a nucleotide sequence.

 <1-3> Homology Analysis of the Inductive Promoter LSIP_P

 The nucleotide sequence of LSIP_P obtained in the above Example <1 # 2> was prepared by Lers et al. C The Journal of Biological Chemistry 1991; 266 (21):

13598-13705) Dunaliella Hamilville revealed in the literature /? Homology was analyzed in comparison with the nucleotide sequence (SEQ ID NO: 14) of the cbr promoter from // e // a bardawi 1).

As a result, the cbr promoter of LSIP_P and D. bardawi 1 of the present invention was found to have a very low homology of 32% (Fig. 2). Example 2

！ _^ ！？ Production of Expression Vectors Including

 In order to confirm the effect of！ ^^ ᅳ cloned in Example 1 and the applicability of the LSIP_P to a heterogeneous organism, Chlamydomonas rain hard tea ((/ a77J¾½70 / S reinhardtin ^ expression system was used.

 In order to use the expression system of λ reinhardtii, pN7-AR_P-LUC-TPsaD, an expression vector for chlamidomonas developed by the present inventors, was used. The pN7—AR_P-LUC-TPsaD is represented by SEQ. It has a nucleotide sequence and consists of a map as shown in FIG. 3.

 <2-1> Cloning of pN7-lLSIPJ-LUC-TPsaD

PCR (Polymerase Chain React ion) using a primer pair of SEQ ID NO: 19 having an Xbal restriction enzyme site and SEQ ID NO: 11 having a restriction enzyme site of EcoRI using pGEM ^(R) -T-LSIP_P prepared in Example 1 as a template ), 1LSIP_P having the nucleus j nucleotide sequence of SEQ ID NO: 18 was obtained.

 Then, pN7 having a map as shown in FIG. 4 by removing AR_P from the pN7-AR_P-LUC-TPsaD using Xbal and EcoRI restriction enzymes and inserting the obtained 1LSIP ᅳ P into the site where the AR_P was removed -lLSIP_P-LUC-TPsaD expression vector was prepared.

 <2-2> Cloning of pN7-4LSIP_P-LUC-TPsaD

PCR (Polymerase Chain React ion) using a primer pair of SEQ ID No. 13 having an Xbal restriction enzyme site and a SEQ ID No. 13 having a restriction enzyme site of EcoRI using pGEM ^(R) -T-LSIP_P prepared in Example 1 as a template ), 4LSIP_P having a nucleotide sequence of SEQ ID NO: 12 was obtained.

Then, by removing the AR_P using the restriction enzymes of Xbal and EcoRI in the pN7-AR_P-LUOTPsaD and inserting the obtained 4LSIP_P into the site where the AR_P was removed, pN7-4LSIP_P-LUC having a map as shown in FIG. A TPsaD expression vector was prepared. <2-3> Cloning of pN7-8LSIP_P-LUC-TPsaD

PCR (Polymerase Chain React ion) using a primer pair of SEQ ID No. 9 having an Xbal restriction enzyme site and SEQ ID NO: 11 having a restriction enzyme site of EcoRI using pGEM ^(R) -T-LSIP_P prepared in Example 1 as a template. ), 8LSIP_P having the nucleotide sequence of SEQ ID NO: 1 was obtained.

 Then, AR_P was removed from the pN7—AR_P-LUC-TPsaD using Xbal and EcoRI restriction enzymes, and the 8LSIP ᅳ P obtained above was inserted into the site where the AR_P was removed, thereby providing a pN7- having a map as shown in FIG. 4. An 8LSIP_P-LUC-TPsaD expression vector was prepared.

 <2-4> Cloning of pN7-17LSIP_P-LUC-TPsaD

PCR (Polymerase Chain React ion) using a primer pair of SEQ ID NO: 10 having an Xbal restriction enzyme site and SEQ ID NO: 11 having a restriction enzyme site of EcoRI using pGEM ^(R) -T-LSIP_P prepared in Example 1 as a template ), 17LSIP_P having the nucleotide sequence of SEQ ID NO: 2 was obtained.

 Then, AR_P was removed from the pN7-AR_P-LUC-TPsaD by using Xbal and EcoRI restriction enzymes, and the 17LSIP_P obtained above was inserted into the site where the AR_P was removed, thereby having pN7-17LSIP_P having a map as shown in FIG. 4. -A LUC-TPsaD expression vector was prepared.

Example 3

 Transformation into Chlai da mas reinhardt ii

 <3-1> Preparation of gamete autolysine

Expression vector pN7-lLSIP prepared in Example 2—P-LUC-TPsaD, _P N7-

In the process of transforming 4LSIP_P-LUC-TPsaD, _P N7-8LSIP_P-LUC-TPsaD, and pN7—17LSIP_P-LUC-TPsaD to C. reinhardt ii, gamete autolysine used for cell wall removal of C. reinhardt ii is conventionally used. Prepared by Harris et al. (Chlamydomonas Handbook (Academic, New York), 00.47, 593-594). Preparation of the g _amet e autolysine To this end, we used wild-type Chlamydomonas reinhardtii CC620 and Chlamydomonas reinhardtii CC621, which are wild type with good mating efficiency, and Wl \ Chlamydomonas reinhardtii CC621. Specifically, each C. reinhardtii CC620 and C. reinhardtii CC621 cells cultured in TAP liquid medium having the composition of Table 1 below were plated on TAP solid medium at a concentration of about 1 × 10 ⁶ cells / plate, and cultured for 1 week. Then, the cultured cells were incubated in TAP liquid medium without a nitrogen source for 5 hours. Then, by counting the respective cell numbers (.reinhardtii CC620 and C. reinhardtii CC621), the cells were mixed to a cell number ratio of 1: 1 and incubated for 15 minutes. C. reinhardtii incubated as described above It was to separate the CC620 and CC621 C. reinhardtii cells wonsam to give the supernatant, by filtering the resulting supernatant with a 0.45 syringe filter was prepared in the gamete autolysine. the gamete autolysine prepared as described above is -80 to -70 ^° Stored in C.

Table 1

<3-2> Introduction of pN7-lLSIP_P-LUC-TPsaD, pN7-4LSIP_P-LUC-TPsaD, pN7-8LSIP_P-LUC-TPsaD and pN7-17LSIP_P-LUC-TPsaD and screening for transformants

Expression vector pN7-lLSIP_P-LUC-TPsaD, pN7- prepared in Example 2, according to a method established in the conventional Kindle et al. (Proc. Natl Acad. Sci. USA 1990; 87: 1228-1232) 4LSIP_P-LUC—TPsaD, _P N7-8LSIP_P-LUC-TPsaD and pN7-17LSIP_P-LUC-TPsaD were introduced into Chlamydoiwnas reinhardtii JL173) cells. Specifically, obtained by centrifugation (.. reinhardtii JL173 cells cultured until the logarithmic growth phase, and resuspended in the gamete autolysine prepared in Example <3-1>, and reacted at room temperature for 60 minutes. After reinhardtii JL173 cells reacted with autolysine were centrifuged again, they were resuspended in TAP liquid medium to adjust the cell concentration to IX 10 ⁸ to 3 × 10 ⁸ cells / ^ Glass beads with diameters of 425 to 600 mm 3 (G8772) 0.3 ml of the cell suspension was dispensed in a test tube containing 0.3 g of (Sigma, USA) .The vector of Example 2 was cut and linearized by 1 / g Kpnl in each test tube. 5 ^ and 100 of 20% PEGCpolyethylene glycol) 8000 was added and vortexed for 25 seconds, and then TAP liquid medium was further added and centrifuged to obtain cells. The cells obtained as described above are resuspended in 10 TAP liquid medium and centrifuged to reacquire the cells. The cells reacquired as above were resuspended in 0.3 ι of TAP liquid medium. Then, add 0.5% agarose, heat and cool to 45 ^° C in TAP medium, add the resuspended cells and mix well. The cells were then hardened by plating on TAP solid medium to which 1 ppm of 3-acetylpyridine (Sigma, USA) was added and incubated for 5-7 days. When the cells cultured for 5 to 7 days start to form green colonies, the colonies were picked with a toothpick and transferred onto a new TAP-prepared medium to express expression vectors pN7-lLSIP_P-LUC-TPsaD, _P N7-4LSIP_P-LUC- C. re / ^ a / ^ // JL173 transformants with TPsaD, pN7-8LSIP—P-LUC-TPsaD and pN7_17LSIP_P-LUC-TPsaD normally transformed were selected.

 <3-3> Confirmation of introduction of pN7-lLSIP_P-LUC-TPsaD, pN7-4LSIP_P-LUC-TPsaD, pN7-8LSIP_P-LUC-TPsaD and pN7-17LSIP— P—LUC-TPsaD

PN7-lLSIP_P-LUC-TPsaD transformed normally selected in Example <3-2>, PN7-4LSIP—P—transformer normally introduced with pC7-7LSIP_P-LUC-TPsaD, pN7-8LSIP_P-LUC-TPsaD Were obtained by culturing the transformants normally introduced with _P N7-17LSIP_P-LUC-TPsaD and culturing them to the logarithmic growth stage in TAP liquid medium, and then DNeasy plant mini kit (69104) (QIAGEN, USA Genomic DNA was isolated using the protocol provided by the manufacturer.

 PN7-lLSIP_P-LUC-TPsaD transformant, pN7-

The transformants in which 4LSIP_P-LUC-TPsaD was normally introduced, the transformants in which pN7-8LSIP_P_LUC-TPsaD was normally introduced, and genomic DNA isolated from pN7-17LSIP_P-LUC-TPsaD were used as templates. PCR was performed with each primer pair to confirm whether a luciferase gene of 936 bp length was introduced.

As a result, a band corresponding to about 936 bp luciferase gene was detected in each g _enom ic DNA (FIG. 5, lanes 1 to 24), and from these results, the vector vector pL LLSIP_P-LUC -TPsaD, pN7-4LSIP_P-LUC- TPsaD, pN7-8LSIP_P-LUC-TPsaD and pNg 17LSIP_P-LUC-TPsaD were confirmed to be normally introduced into (. Reinhardtii JL173 cells.

Example 4

！ _^ ^！？ Activity measurement

PN7-lLSIP_P-LUC-TPsaD transformant, PN7-4LSIP_P-LUC-TPsaD transformant, pN7-8LSIP_P-LUOTPsaD transformant and PN7-17LSIP_P-LUC-TPsaD transformant selected in Example <3-2> above The converters each contain a TAP liquid medium While dividing and incubating in three test tubes, the test tubes were dark-treated or irradiated with light of 30, 300 or 600 umol photon / m ² / s for about 3 hours. Then, the transformant was obtained, and lucifer with Luminometer (GloMax ^TM 20/20) (Pr omega, USA) according to the protocol provided by the manufacturer using Renilla Lucif erase assay kit (E2810) (Promega, USA). The amount of luminescence of luciferase was measured. In order to analyze the degree of luciferase expression at each intensity, the luminescence of the transformant treated with the luminescence of the transformant irradiated with light of 0, 30, 300 or 600 umol photon / mVs The luminescence ratio was compared by dividing by (luminescence).

As a result, the reporter gene luciferase showed high luminescence regardless of the light intensity irradiated downstream of 1LSIP_P. In addition, luciferase, a reporter gene, showed higher luminescence as the intensity of light irradiated downstream of 4LSIP_P, 8LSIP_P and 17LSIP_P increased, with weak intensity of light (0 or 30 umol _P hoton / m ² / s) and strong The difference in the emitted luminescence ratio was large in the light of intensity (300 or 600 umol photon / m ² / s) (Tables 2, 6 and 7). From the above results, it can be seen that 1LSIP 'P having the nucleotide sequence of SEQ ID NO: 18 is a basic unit for LSIP_P of the present invention to have promoter activity. Accordingly, the LSIP_P of the present invention, such as 4LSIP_P, 8LSIP_P, or 17LSIP_P, in which some nucleotide sequences are added at the 5 'end of the 1LSIP_P, regulates the expression of a gene linked downstream thereof depending on the light intensity to be irradiated. It can be seen.

Table 2

Example 5

 Activity Comparison with Dunaliella bambil-derived cbr promoter

 Lers et al. (The Journal of Biological Chemistry 1991; 266 (21): 13598-13705) Dunaliella Bambil 0 ^ // e // a bar daw m derived cund promoter and Dunaliella sp of the present invention. The derived LSIP promoters were compared for their ability to induce promoter activity by light.

Lers et al. PCR (Polymerase) was carried out using primer pairs of SEQ ID NO: 16 having an Xbal restriction enzyme site and SEQ ID NO: 17 having a restriction enzyme site of EcoRI, using the genomic DNA of Dunaliella thabil established in the literature as a template. Chain Reaction) gave 831-081? _1 ³ having the nucleotide sequence of SEQ ID NO.

 Then, in the same manner as in Example 2 by removing the AR ᅳ P in the pN7-AR_P-LUC-TPsaD using Xbal and EcoRI restriction enzymes, by inserting the Bar-CBR_P obtained in the site where the AR_P is removed, PN7-Bar-CBR_P-LUC-TPsaD expression vector having a map as shown in Figure 4 was prepared.

 In the same manner as in Example 3 (p. Barg-CBR_P-LUC-TPsaD expression vector prepared as described above); transformed with reinhardtii, and screened for transformants transformed properly, and then in the transformant PCR was performed using the isolated pN7-Bar-CBR ᅳ P-LUC-TPsaD genomic DNA as a template and primer pairs of SEQ ID NO: 20 and SEQ ID NO: 21 to identify the band corresponding to the 936 bp luciferase gene (Fig. 5, lanes 25 to 30), it was confirmed that the pNg Bar-CBR_P-LUC-TPsaD was normally introduced into C. reinhardtii JL173 cells.

PN7-Bar-CBR_P-LUOTPsaD transformant screened as described above, pN7-lLSIPLS P-LUC-TPsaD transformant screened in Example <3-2>, pN7-4LSIP_P-LUC-TPsaD transformant treatment of the test tubes with cancer or 30 or 300 by dividing the pN7-8LSIP_P-LUC-TPsaD transformant and the pN 17LSIP ᅳ P-LUC-TPsaD transformant into several test tubes each containing a TAP liquid medium. The light of ymol photon / m ² / s was irradiated for about 3 hours. Then, the transformant was obtained and lucifered with Luminometer (GloMax ™ 20/20) (Promega, USA) according to the protocol provided by the manufacturer using Renilla Luc if erase assay kit (E2810) (Promega, USA). The amount of luminescence of luciferase was measured. In order to analyze the degree of luciferase expression at each intensity, the luminescence of the transformants irradiated with light of 300 umol photon / m ² / s was measured for 30 ymol photon / m ² / s of the transformants. The luminescence ratio was compared by dividing by luminescence.

As a result, the reporter gene luciferase showed a more stable and constant expression pattern under the 1LSIP_P of the present invention than under the Dunaliella hambil-derived cbr promoter (Table 3 and FIG. 6). In other words, cinna promoter derived from Dunaliella haebil showed a large variation in promoter activity for each clone. The 1LSIP_P promoter was found to exhibit a constant level of promoter activity per clone. In addition, the reporter gene luciferase than Dunalielli sp. Higher luminescence ratios were shown under derived LSIP_P (4LSIP_P, 8LSIP_P and 17LSIIP_P) (Table 3 and FIG. 7). This is Dunaliella Esp. Derived LSIP_P reacted more sensitively to the light intensity irradiated than Dunaliella hamvil derived cbr promoter, thereby regulating the expression of luciferase.

 From the above results, LSIP_P of the present invention is based on the base of 1LSIP_P having a nucleotide sequence of SEQ ID NO: 18, which shows a more stable promoter activity than that of the Dunaliella hamville-derived cbr promoter. It can be seen that it is more sensitive to light intensity than a promoter and regulates the expression of genes linked downstream more sensitively. Table 3

상기에서는 본 발명의 바람직한 실시예를 예시적으로 설명하였으나， 본 발명의 범위는 상기와 같은 특정 실시예에만 한정되지 아나하며， 해당 분야에서 통상의 지식을 가진 자라면 본 발명의 특허청구범위에 기재된 범주 내에서 적절하게 변경이 가능할 것이다.

In the above description of the preferred embodiment of the present invention by way of example, the scope of the present invention is not limited to the specific embodiment as described above, those skilled in the art described in the claims of the present invention Changes may be made as appropriate within the scope.

Claims

[Patent Claims] [Claim 1]  A light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12. [Claim 2]  The light inducible promoter according to claim 1, wherein the promoter comprises a nucleotide sequence set forth in SEQ ID NO: 1. [Claim 3]  The light inducible promoter according to claim 1, wherein the promoter comprises a nucleotide sequence set forth in SEQ ID NO: 2. [Claim 4]  An expression vector comprising the light inducible promoter of claim 1. [Claim 5]  The expression vector of claim 4, wherein the expression vector further comprises a multiple cloning site (MCS) through which a nucleotide sequence encoding a foreign protein can be operably inserted into the promoter. [Claim 6]  A transformant transformed with the expression vector of claim 4. [Claim 7] An expression construct in which a nucleotide sequence encoding a foreign protein is operably linked to a light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12. [Claim 8]  An expression vector comprising the expression construct of claim 7. [Claim 9]  A transformant transformed with the expression vector of claim 8. [Claim 10]  Method of producing foreign protein comprising the following steps:  (a) the transformant of claim 6 or 9 transformed by an expression vector comprising a nucleotide sequence encoding a foreign protein operably linked to a light inducible promoter comprising the nucleotide sequence set forth in SEQ ID NO: 12 Culturing; And  (b) irradiating the cultured transformant with light to produce the foreign protein.