KR20190047914A - Recombinant vector for microalgae transformation comprising sequence derived from nitrate reductase and uses thereof - Google Patents

Abstract

The present invention relates to a microalgae transformation recombinant vector characterized in that a nitrate reductase N-terminal coding polynucleotide, a promoter, multiple cloning sites (MCSs) for inserting foreign genes, a terminator, and a nitrate reductase C-terminal coding polynucleotide are operatively connected in the 5′ to 3′ direction, and a use thereof.

Description

Recombinant vector for transforming microalgae containing a nitrate reductase-derived sequence and use thereof [0002] Recombinant vector for microalgae transformation derived from nitrate reductase and uses thereof [

The present invention relates to a recombinant vector for transforming microalgae containing a nitrate reductase-derived sequence and use thereof.

The market for recombinant proteins has grown 35% annually since 2001 and is projected to grow by 8.83% annually from 2013 to 2018, reaching $ 141 billion by 2017. The recombinant proteins currently being used are 39% Escherichia coli , 35% Chinese hamster ovary (CHO) cells, 15% yeast, and 10% other mammalian systems. The production of various therapeutic proteins and vaccines using transgenic microalgae has been carried out, and it is expected that the production of recombinant proteins using microalgae will be increased in the future due to various advantages of microalgae.

Transformation techniques of microalgae have been developed using Chlamydomonas reinhardtii as a representative model, but recently, transformation techniques targeting various microalgae such as Chlorella have been reported. The inventors of the present invention have previously carried out a method of introducing a growth hormone of a flounder into a microalgae, Chlorella ellipsoidea , and then transforming them into Brine shrimp Artemia, And the growth of the flounder was increased by 25% compared to the control.

The problem with the microalgae transformation technology that is currently being developed is that several genes are introduced into the genome of the microalgae according to the amount of the plasmid used for transfection so that the mutation of the original gene can be induced . A more important problem is that the method of effectively screening transgenic plants is very limited, costly and environmentally hazardous. That is, in order to select a transformant, generally, an antibiotic resistance gene is introduced simultaneously with a gene to be introduced, and a transformant is selected from a medium containing the antibiotic. However, since microalgae are eukaryotic organisms, they have a natural tolerance to most antibiotics. Therefore, antibiotics that can be used for screening transformants are very limited. There is also a difference in antibiotic resistance between microalgae and antibiotics Its use is limited because it is very expensive. For example, a transformant with a ble gene can be screened using bleomycin, but the price of 50 mg of bleomycin is about 1,374,000 won.

Accordingly, the present inventors have developed a recombinant vector containing part of the DNA sequence of a microalgae-derived nitrate reductase, introduced a foreign gene through homologous recombination through the DNA sequence of the nitrate reductase, In a medium containing chlorate rather than an antibiotic.

Korean Patent Publication No. 2011-0090565 discloses 'a vector useful for transformation of microalgae and microalgae transformed with the vector', and Korean Patent No. 1567308 discloses 'microalgae biomass and microalgae' A recombinant vector for increasing lipid productivity and its use "has been disclosed. However, the recombinant vector for transforming microalgae containing the nitrate reductase-derived sequence of the present invention and its use have not been described.

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to provide a recombinant vector in which N-terminal and C-terminal sequences of chlorella-derived nitrate reductase coding gene are respectively linked upstream of a promoter and downstream of a terminator, A recombinant vector into which a gene coding for glycoprotein of hemorrhagic sepsis virus (VHSV) was introduced was transformed into a chlorella bulgaris strain and then cultured in a selective medium containing potassium chlorate. As a result, a glycoprotein coding gene of VHSV was introduced Confirming that only the transformant grows in the above selection medium, thereby completing the present invention.

In order to solve the above-described problems, the present invention provides an N-terminal coding polynucleotide of nitrate reductase, a promoter, an MCS (multiple cloning site) for foreign gene insertion, a terminator and a C- End-coding polynucleotide is operably linked to a recombinant vector for microalgae transformation.

The present invention also provides a microalgae transformed with a recombinant vector of a foreign gene into which the foreign gene is inserted into the MCS of the recombinant vector.

The present invention also provides a method for producing a recombinant vector, comprising: preparing a recombinant vector of a foreign gene by inserting a foreign gene into the recombinant vector; Transforming the microalgae cells with a recombinant vector of the foreign gene; And culturing the transformed microalgae in a selective medium containing chlorate. The present invention also provides a method for screening transformed microalgae.

The microalgae transformation method of the present invention can effectively introduce a foreign gene through homologous recombination between a DNA fragment derived from a nitrate reductase gene in a recombinant vector and a nitrate reductase gene on a genome, and the homologous recombination can produce a nitrate reductase gene Is replaced with a foreign gene and the nitrate reductase does not function, the transformed microalgae can survive in the medium containing chlorate, so that the transformant can be easily selected. The recombinant vector of the present invention and the method of transforming microalgae using the recombinant vector of the present invention are an economical and easy method to overcome the problems of the conventional microalgae transformation and screening of transformants.

1 is a schematic diagram of microalgae transformation using the recombinant vector of the present invention.
2 is a schematic diagram of the recombinant vector pSK-HR-VHSVG of the present invention.
FIG. 3 shows the results of confirming the growth of the strain in media containing 0, 50, 100 and 150 mM potassium chlorate to confirm the resistance of the wild-type chlorella bulgurris PKVL7422 to potassium chlorate.
FIG. 4 shows the results of confirming the characteristics of the wild type chlorella bulgaris PKVL7422 in the potassium chlorate-containing medium in order to find the transformant selection medium. In FIG. 4, A shows BG-11 medium containing no light + potassium chlorate, , BG-11 medium lacking glucose and potassium chlorate, medium C containing light + 0.5% glucose and BG-11 medium containing 150 mM potassium chlorate, D light-free condition + 0.5% glucose and 150 mM potassium chlorate BG-11 medium, and the circle inserted in each picture means the picture from the front of the plate culture medium.
FIG. 5 shows the result of screening of transformants. As a result of screening of the transformants, it was confirmed that chlorella bulgaris PKVL7422 (A) not subjected to electroporation and chlorella bulgurris PKVL7422 (B) after electroporation using the vector shown in FIG. 14 days after culturing.
FIG. 6 shows the result of PCR to confirm whether or not the transgene was selected from a randomly selected transformant. M: size marker, P: vector DNA used for transformation, N: wild-type Chlorella vulgaris, 1-6: randomly selected transformed Chlorella vulgaris.
7 is a growth curve of the transformed chlorella bulgurris PKVL7422 in the presence of 150 mM potassium chlorate.
FIG. 8 shows the result of confirming the introduction of a foreign gene through a polymerase chain reaction. M: size marker, P: vector DNA used for transformation, N: wild-type Chlorella vulgaris, T: transformed Chlorella vulgaris.
Fig. 9 shows the result of confirming expression of a target protein using Western blotting. M: size marker, P: purely isolated VHSV glycoprotein (identified by two bands due to differences in glycation), N: total protein extracted from wild type chlorella bulgaris, T: whole protein extracted from transformed chlorella bulguris.

In order to accomplish the object of the present invention, the present invention provides an N-terminal coding polynucleotide of nitrate reductase, a promoter, an MCS (multiple cloning site) for inserting a foreign gene, a terminator and a nitrate reductase Wherein the C-terminal coding polynucleotide is operably linked to the recombinant vector.

In the recombinant vector of the present invention, the nitrate reductase may be a derived from Chlorella, preferably chlorella Barrier borrowed switch (Chlorella variabilis , < / RTI > but are not limited thereto.

In a recombinant vector for transformation of microalgae, according to one embodiment of the invention, the microalgae include, but are not limited to, Chlorella vulgaris (Chlorella vulgaris), Chlorella no other trad (C. anitrata), Chlorella hits arcs urticae (C. antarctica , C. aureoviridis , C. candida , C. capsulate , C. desiccate , C. ellipsoidea , C. elegans , Chlorella such as C. emersonii and C. fusca , Spirulina, Nannochloropsis, Tetraselmis, Cheaoceros, Iso Such as Isochryosis, Pavlova, Phaeodactylum, Skeletonema, Navicula, Calonase, and the like.

In the recombinant vector of the present invention, the N-terminal coding polynucleotide and the C-terminal coding polynucleotide of the nitrate reductase are sufficiently large enough to allow homologous recombination with the nitrate reductase coding gene in the microalgae, But are not limited to, the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. The homologue is a base sequence having the same functional characteristics as the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, although the nucleotide sequence thereof is changed. Specifically, the N-terminal coding polynucleotide and the C-terminal coding polynucleotide of the nitrate reductase are respectively at least 70%, more preferably at least 80%, even more preferably at least 70% May comprise a nucleotide sequence having a sequence homology of 90% or more, and most preferably 95% or more. &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The term " recombinant " refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. Recombinant cells can express a gene or a gene fragment that is not found in the natural form of the cell, either in a sense or in an antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term " vector " is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term " carrier " is often used interchangeably with " vector ".

The recombinant vector of the present invention may contain a promoter. The promoter is preferably a CaMV 35S promoter (SEQ ID NO: 3), an actin promoter, an ubiquitin promoter, a pEMU promoter, a MAS promoter or a histone promoter, more preferably a CaMV 35S promoter , But is not limited thereto. The term " promoter " refers to a region upstream of DNA from a structural gene, and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.

The recombinant vector of the present invention may include a terminator under the MCS. The terminator can be a conventional terminator, such as a Ribs-1,5-bisphosphate carboxylase / oxygenase (RbcS2) terminator (SEQ ID NO: 4), a nopaline synthase (NOS) terminator, rice α- amylase RAmy1 A terminator A phaseoline terminator, and a terminator of the Octopine gene of Agrobacterium tumefaciens , but are not limited thereto.

The term " MCS (multiple cloning site) " refers to a DNA fragment having a variety of restriction enzyme sites, which is recognized as a specific restriction enzyme and thus is capable of insertion of the foreign gene of interest into the region of the cleaved MCS. MCSs that can be used in the vectors of the present invention can be used without limitation as long as they are MCS known in the art, which can be obtained from various vectors known in the art having MCS. In a recombinant vector according to an embodiment of the present invention, the MCS may include BamH I and Xho I restriction sites in the 5 'to 3' direction, but is not limited thereto.

The term " operably linked " in the present invention means that one nucleic acid fragment is associated with another nucleic acid fragment so that its function or expression is affected by other nucleic acid fragments. That is, the gene coding for the exogenous protein can be linked so that its expression can be regulated by a promoter in the vector.

The recombinant vector according to the present invention does not contain a selectable marker consisting of a nucleic acid sequence having characteristics that can be selected by conventional chemical methods in order to distinguish the transformed cells from non-transformed cells.

The present invention also provides a microalgae transformed with a recombinant vector of a foreign gene into which the foreign gene is inserted into the MCS of the recombinant vector.

The foreign gene of the present invention may be a gene encoding a target protein to be expressed in a microalgae, and may be, for example, a pharmaceutical protein coding gene, a vaccine antigen protein coding gene, and the like.

The transformed microalgae according to the present invention include, but are not limited to, Chlorella vulgaris), Chlorella No other Trapani (C. anitrata), Chlorella hits Kirk Utica (C. antarctica), Chlorella brother Leo corruption disk (C. aureoviridis), Chlorella Candida (C. candida), Chlorella encapsulated (C. capsulate) Chlorella such as C. desiccate , C. ellipsoidea , C. emersonii and C. fusca , Spirulina, nano-chlorophenol, For example, Nannochloropsis, Tetraselmis, Cheaoceros, Isochryosis, Pavlova, Phaeodactylum, Skeletonema, Navicula, , Calonase, and the like.

The present invention also relates to

Inserting a foreign gene into the recombinant vector to produce a recombinant vector of the foreign gene;

Transforming the microalgae cells with a recombinant vector of the foreign gene; And

And culturing the transformed microalgae in a selection medium containing chlorate. The present invention also provides a method for screening transformed microalgae.

In the screening method of the present invention, transformation methods for introducing a recombinant vector into microalgae include electroporation, glass bead, gene gun, silicon carbide whisker, Or the like, and preferably, it may be an electroporation method, but is not limited thereto.

In the screening method of the present invention, the chlorate may be potassium chlorate, sodium chlorate, lithium chlorate or the like, preferably potassium chlorate, but is not limited thereto. The concentration of potassium chlorate in the selection medium may be, for example, 120 to 180 mM, preferably 140 to 160 mM, more preferably 150 mM, but is not limited thereto.

In addition, the selection medium of the present invention may further include glucose, and the concentration of added glucose may be 0.4 to 0.6% (w / v), but is not limited thereto.

In the screening method of the present invention, the transformed microalgae may be selected from the group consisting of an N-terminal coding polynucleotide of nitrate reductase and a C-terminal coding polynucleotide sequence of nitrate reductase in the recombinant vector, and a genomic nitrate reductase coding gene The foreign gene is introduced by homologous recombination, the nitrate reductase gene in the genome is defective, and the nitrate reductase is a microalga that has lost its function. The nitrate reductase has the function of reducing the nitrate ion (NO ₃ ^- ) to the nitrite ion (NO ₂ ^- ), but also the chlorate ion (ClO ₃ ^- ) to the toxic chlorine dioxide (ClO ₂ ^- ). ≪ / RTI > Therefore, the transformed microalgae of the present invention can not reduce chlorate ion to chlorite ion because the nitrate reductase does not function, so that it is resistant to the culture solution containing chlorate ion, , It is possible to grow in a culture medium containing chlorate. On the other hand, untransformed microalgae can function as a nitrate reductase, so that chlorate ions can be reduced to chlorite ions, which makes growth impossible in a culture medium containing chlorate ions. Therefore, the transformant and the non-transformant can be selected only by the addition of the chlorate ion in the culture solution, so that no separate antibiotic is required.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

1. Construction of Transformation Vector

In the present invention, the vector used for microalgae transformation was prepared based on pBluescript SK (+), a bacterial transformation vector, and a schematic diagram of the constructed vector is shown in FIG. The vector of the present invention can be used for transforming Chlorella species in microalgae , such as Chlorella variabilis The DNA fragment of NC64A nitrate reductase (NR) was arranged at both ends of the gene to be inserted. The gene to be inserted was the glycoprotein of the viral hemorrhagic septicemia virus (VHSV) glycoprotein gene was used and the 35S promoter of CaMV (cauliflower mosaic virus) required for expression of the gene and the transcription termination of Ribs-1,5-bisphosphate carboxylase / oxygenase (RbcS2) gene of Chlamydomonas reinhardtii And a transcription terminator.

The NR gene DNA fragment, chlorella Varia NR genetic information of Billy's NC64A: Based on the (GenBank Accession number CHLNCD RAFT_56304) was synthesized in ㈜ Bioneer (South Korea), NR gene segment which is located in front of the CaMV 35S promoter ( 5 'fragment NR) corresponds to 1 to 254 th nucleotide in the gene, the 5' end was inserted into the 3 'Apa I, respectively at the ends (GGGCCC) with EcoR 1 (GAATTC) restriction enzyme sequence. The NR gene fragment (3 'NR fragment) located at the rear of the transcription termini of the rbcs2 gene corresponds to the 2,486-2,589th nucleotide of the gene, and Not I (GCGGCCGC) and Nde 1 ( CATATG) restriction enzyme sequence was inserted.

The inserted VHSV glycoprotein coding gene was synthesized on the basis of the base sequence of the VHSV genome (GenBank accession number: JQ651388.1), and corresponds to the 1-1560 base sequence of the above-mentioned nucleotide sequence. In order to increase the expression efficiency BamH 1 (GGATCC) and Xho 1 (CTCGAG) restriction enzyme sequences were inserted for efficient cloning in order to optimize the codons of Chlorella vulgaris .

The vector thus constructed was named pSK-NR-VHSVG.

2. Microalgae and culture

The microalgae used in the transformation were Chlorella vulgaris strain PKVL7422 (accession number: KCTC13361BP) and cultured at a temperature of 20 ° C in BG-11 medium. The composition of BG-11 medium was as shown in Table 1 Respectively.

3. Selection of transformants and transformants

The strain of Chlorella vulgaris PKVL7422 was cultured in BG-11 medium for about 15 days so that the cell number became 1.0 × 10 ⁷ cells / ml. Then, 30 ml of the strain was centrifuged at 2,700 × g at 4 ° C. for 45 minutes. After centrifugation, the supernatant was removed, and 1 ml of cell preservation solution (0.2 M mannitol, 0.2 M sorbitol) was added to the pellet, followed by incubation at room temperature for 1 hour. After centrifugation at 3,300 x g at 4 ° C for 10 minutes, the supernatant was removed, and the cell pellet was subjected to electrophoresis (500 mM NaCl, 5 mM KCl, 5 mM CaCl ₂ , 20 mM Hepes, 200 mM mannitol, 200 mM sorbitol; pH 7.2) was added and mixed. Then, 20 μg of the vector DNA prepared above was added, and the mixture was kept on ice for 10 minutes. 1 ml of the cell solution containing the vector DNA was transferred to an electrospray cuvette and electroporated for 3 to 5 seconds at 1.00 kV and 400 ohm. 1 ml of the perforated cell solution was mixed with 5 ml of BG-11 medium and incubated overnight at 20 ° C in a dark condition to restore the cells. 200 μl of the recovered cell solution was spread evenly on a modified BG-11 (NH4) medium containing 150 mM potassium chlorate (KClO ₃ ), 0.5% glucose and 17 mM ammonium ion (NH ₄ ⁺ ), After wrapping with foil or the like, the cells were cultured at 20 ° C for 15 days. Modified medium BG-11 (NH ₄ ⁺⁾ is removed by NR yujeonga is introduced when the microalgae transgene nitrate ion (NO ₃ ^-) a nitrite ^ion due to not be reduced to be used as sources of nitrogen (NO ₂₎ It contains ammonium chloride (NH ₄ Cl) as the nitrogen source. In addition, 0.5% glucose was contained for culturing in a dark state, and the composition of the modified BG-11 (NH4) medium is shown in Table 2 below.

Compositions for selection of transformants BG11 (NH4) 150mM KClO ₃ Glucose medium (1L) Stock 1 (see Table 1) 10 ml Stock 2 (see Table 1) 10 ml Stock 3 (see Table 1) 10 ml Na ₂ CO ₃ 0.02 g Stock 5 (see Table 1) 1.0 ml NH ₄ Cl 0.9 g KClO ₃ 18.3 g Glucose 5 g Agar 7 g After sterilization at high pressure, set it to pH 7.1. Pour 25 ~ 30 ml per plate and use after hardening.

4. Selection and characterization of transformants

1) Selection of transformants

Transformants were selected by confirming the formation of colonies after culturing in the selection medium at 20 ° C for 15 days. Each colony was judged to be derived from the respective transformed Chlorella vulgaris and was assigned a unique strain number.

2) Culturing the transformant

Chlorella vulgaris colonies grew on the above selection medium were re-inoculated into BG-11 (NH4) liquid medium and cultured at 20 DEG C for 15 days.

3) Polymerase chain reaction

DNA for PCR was extracted from transformed chlorella and control using gene extraction reagent of Biona Co., Ltd. 1 ml of chlorella culture grown at a concentration of about 1.0 × 10 ⁷ cells / ml was centrifuged at 3,300 × g for 10 minutes, and the supernatant was removed. The resulting cell pellet was used for DNA extraction. The extracted DNA was quantified using nano-drop and stored at -20 ° C. PCR was performed using AmpONE HS taq premix kit from Genell (Korea). 1 μl of the GF primer (5'-GCACCACACCACAGATCACC-3 '; SEQ ID NO: 5) and the GR primer (5'-ACTTCCGCGAGTAGAGGTCA-3'; SEQ ID NO: 6), 1 μl of the extracted DNA, And then PCR was carried out. The PCR amplification reaction was performed by denaturing at 94 ° C for 4 minutes, followed by 30 reactions at 94 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 1 minute and 30 seconds, followed by reaction at 74 ° C for 10 minutes. Were electrophoresed on 1% agarose gel and examined. The two primers used in the PCR amplify a portion of the VHSV glycoprotein coding gene and are 495 bp in size.

4) induction of protein expression from transgene

30 ml of the transformed chlorella culture solution at 1 × 10 ⁷ cells / ml was centrifuged at 2,700 × g for 45 minutes, and the supernatant was completely removed. Then, protein extraction buffer (Ripa buffer 800 μl, ELPIS BIOTECH, Korea), protease inhibitor cocktail 200 Mu l), and the mixture was transferred into a 1.5-ml tube. The mixture was kept on ice for 30 minutes, centrifuged at 3,300 x g for 10 minutes, and the supernatant was transferred to a new tube. 100 μl of a 5 × SDS sample loading buffer was added thereto, boiled at 90 ° C. for 10 minutes, and then kept on ice for 5 minutes. The proteins were separated by electrophoresis on 12% SDS PAGE. The separated proteins were transferred to a PVDF membrane under the condition of 50 V for 3 hours. Then, the polyclonal antibody produced in rabbit was used as a primary antibody and AP (alkaline phosphate) was conjugated with VHSV glycoprotein expressed in Escherichia coli rabbit anti-mouse IgG (Sigma, USA) as a secondary antibody for 1 hour each. After the reaction, the membrane was washed with PBST (1 × PBS + 0.05% Tween 20) three times for 10 minutes, and then protein expression was confirmed by detection reagent (Alkaline phosphatase reaction buffer 30 ml, NBT 198 μl, BCIP 99 μl).

Example 1 Growth Analysis of Chlorella virulis PKVL7422 Strain in the Presence of Potassium Chlorate

A feature of the present invention is that the transformed cells are selected by using potassium chlorate (KClO ₃ ) by replacing the NR gene of chlorella bulgaris with an introduced gene. Therefore, the resistance to the potassium chlorate of the wild type chlorella bulguris PKVL7422, which is not transformed, is an important factor. As shown in FIG. 3, when wild type chlorella bulgur PKVL7422 was cultured in BG-11 medium containing 0, 50, 100 and 150 mM potassium chlorate, it was confirmed that the strain did not grow at a concentration of 100 mM or more, The use of the medium containing potassium chlorate as above means that the transformants can be selected.

Example 2. Culture of chlorella bulgaris PKVL7422 strain and screening of transformants

In order to mass-culture the transformant in a fermenter after selecting the transformant, the transformant should be able to grow using the carbon source contained in the medium without photosynthesis. Fig. 4A shows the results obtained when the strain of Chlorella vulgaris PKVL7422 was inoculated into BG-11 medium and incubated under light conditions. Fig. 4B shows the result of inoculation of Chlorella vulgaris strain PKVL7422 into BG-11 medium supplemented with 0.5% It's a picture. From these results, it was confirmed that the chlorella bulgurris strain PKVL7422 used in the present invention can grow well under cancer conditions when the carbon source is appropriate. In order to establish selection conditions for transformants using potassium chlorate, wild type chlorella bulgaris PKVL7422 strain was inoculated into BG-11 (NH4) medium containing 150 mM potassium chlorate and incubated under light conditions. However, As a result, only a small number of colonies were formed as well as cells showing resistance to chlorate ion (ClO ₂ ^- ) by natural mutation. In order to solve the above problem, wild type chlorella bulgurris PKVL7422 strain was inoculated into BG-11 (NH4) medium containing 150 mM potassium chlorate and 5% glucose, and cultured under the dark condition as shown in FIG. 4D, It was confirmed that culturing the cells in BG-11 (NH4) medium containing 150 mM potassium chlorate and 0.5% glucose was appropriate for selecting the transformed individuals.

Based on the above results, the cells transformed by electroporation were plated on BG-11 (NH4) medium containing 150 mM potassium chlorate and 0.5% glucose, wrapped in aluminum foil to prevent light from entering, As a result, colonies were formed as shown in FIG. 5, and no growth was observed in the wild type chlorella bulgaris cells that had not been transformed, and it was confirmed that the transformants were selected. In addition, the culture of chlorella bulgaris formed in the transformant selection medium was arbitrarily selected and cultured. Then, DNA was extracted by the above-mentioned method, and PCR was performed using GF and GR primers. As a result, The DNA to be introduced was amplified (FIG. 6) and confirmed to be a highly efficient transformation method with a transformation ratio of 100%.

Example 3. Analysis of growth characteristics of transformants

As shown in FIG. 7, the growth characteristics of the transformed microalgae were slowly grown for 7 days in BG-11 (NH4) medium containing 150 mM potassium chlorate and 0.5% glucose, And the degree of its growth was found to be one third of the wild type in BG-11 medium containing 0.5% glucose without potassium chlorate. On the other hand, wild type did not grow in BG-11 (NH4) medium containing 150 mM potassium chlorate and 0.5% glucose.

Example 4. Analysis of Insertion Gene and Protein Expression of Transformants

4-1. Identification of glycoprotein gene derived from VHSV in transformed microalgae by polymerase chain reaction

In order to determine whether the desired DNA was inserted into the chromosomal DNA of the microalgae from the vector DNA which was introduced into the microalgae through electric perforation, a primer targeting a part of the VHSV-derived glycoprotein gene contained in the vector was used for polymerase chain reaction Respectively. As a result, as shown in Fig. 8, the amplified product was not detected in the wild type chlorella without transformation, but in the positive control DNA of the transformed chlorella derived from the colonies grown in the selection medium, the expected size DNA was amplified, It was confirmed that the desired DNA was inserted into the chromosome of chlorella by homologous recombination.

4-2. Confirmation of expression of VHSV-derived glycoprotein in transgenic microalgae via Western blotting

The expression of the target protein was confirmed by Western blotting from the transformed microalgae in which the insertion of the target DNA was confirmed through the polymerase chain reaction. The whole protein was extracted from wild type and transformed chlorella bulgur PKVL7422 and subjected to Western blotting according to the method described above after electrophoresis. As a result, the protein band reacting to the VHSV glycoprotein antibody shown in Fig. 9 was not confirmed in the wild type microalgae But not in transgenic microalgae. These results show that the desired DNA is inserted into the chromosomal DNA of the microalgae and the desired protein is expressed therefrom. Thus, the microalgae can be efficiently transformed by the microalgae transformation method of the present invention, and the desired recombinant protein Which means that it can produce.

<110> Pukyong National University Industry-University Cooperation Foundation <120> Recombinant vector for microalgae transformation comprising          sequence derived from nitrate reductase and uses thereof <130> PN17404 <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 254 <212> DNA <213> Chlorella variabilis <400> 1 atgaccgtcc tcctcaacgc cgacgactct gcccacggca gcagctcggc agcgacggtc 60 atcaaagcga atggacactg cccggctgca gcagggaatg gggcgccggc accagccctg 120 cccgcagccc ccgcctgcgg cccgccgccc cagcagcccc cgctggagcc cggcaccgag 180 gattacaagc tccacctgcc agtgtccgag gtgctggact tcgacaaggg cacaaaggat 240 gactgggtgc cacg 254 <210> 2 <211> 254 <212> DNA <213> Chlorella variabilis <400> 2 acaccatctg ctgcctgtgc ggcccacccc ccatgatcaa gtttgcctgc ctgccggtga 60 ggccccgccc gcagcaggca tccctgctgg ctgcccccct gctgctgccg cccgagccgc 120 tgccggcagg ctggggaacc cacggagcat gctaagtact gctgttgttt ttgagtcgtc 180 ctgcctgcct gcctgtccgc ctgcagaacc tggagaagct tggctacaag ccggagcagt 240 gcatccagtt ctga 254 <210> 3 <211> 892 <212> DNA <213> Cauliflower mosaic virus <400> 3 ggtgctgcta tcgataccgt cgaagcttgc atgcctgcag gtccccagat tagccttttc 60 aatttcagaa agaatgctaa cccacagatg gttagagagg cttacgcagc aggtctcatc 120 aagacgatct acccgagcaa taatctccag gaaatcaaat accttcccaa gaaggttaaa 180 gatgcagtca aaagattcag gactaactgc atcaagaaca cagagaaaga tatatttctc 240 aagatcagaa gtactattcc agtatggacg attcaaggct tgcttcacaa accaaggcaa 300 gtaatagaga ttggagtctc taaaaaggta gttcccactg aatcaaaggc catggagtca 360 aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca gttcatacag 420 agtctcttcg actcaatgac aagaagaaaa tcttcgtcaa catggtggag cacgacacac 480 ttgtctactc caaaaatatc aaagatacaga tctcagaaga ccaaagggca attgagactt 540 ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 600 ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 660 gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 720 cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 780 gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 840 cctctatata aggaagttca tttcatttgg agagaacacg ggggactcta ga 892 <210> 4 <211> 284 <212> DNA <213> Chlamydomonas reinhardtii <400> 4 ccgacgtcga gccactctag aagatccccg ctccgtgtaa atggaggcgc tcgttgatct 60 gagccttgcc ccctgacgaa cggcggtgga tggagagtac tgctctcaag tgctgaagcg 120 gtagcttagc tccccgtttc gtgctgatca gtctttttca acacgtaaaa agcggaggag 180 ttttgcaatt ttgttggttg taacgatcct ccgttgattt tggcctcttt ctccatgggc 240 gggctgggcg tatttgaagc gggtaccgac ctcgaattgt cgag 284 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gcaccacatc acagatcacc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acttccgcga gtagaggtca 20

Claims (8)

Terminal coding polynucleotide of nitrate reductase, a promoter, a multiple cloning site (MCS) for foreign gene insertion, a terminator and a C-terminal coding polynucleotide of nitrate reductase operably linked in the 5 'to 3' direction A recombinant vector for transforming microalgae. 2. The micro-algae transformation vector according to claim 1, wherein the N-terminal coding polynucleotide of the nitrate reductase is the nucleotide sequence of SEQ ID NO: 1. 2. The micro-algae transformation vector according to claim 1, wherein the C-terminal coding polynucleotide of the nitrate reductase is the nucleotide sequence of SEQ ID NO: 2. 2. The microalgae-transforming recombinant vector according to claim 1, wherein the foreign gene is inserted into the MCS. A microalgae transformed with the recombinant vector of any one of claims 1 to 4. Preparing a recombinant vector of a foreign gene by inserting a foreign gene into the recombinant vector of any one of claims 1 to 3;
Transforming the microalgae cells with a recombinant vector of the foreign gene; And
Culturing the transformed microalgae in a selection medium containing chlorate, and selecting the transformed microalgae. 7. The method according to claim 6, wherein the selection medium further comprises glucose. 7. The method according to claim 6, wherein the chlorate is potassium chlorate, sodium chlorate or lithium chlorate.

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