KR20180038270A - Novel method for gene expression in Thraustochytrid microalgae - Google Patents

Abstract

The present invention relates to a method for overexpressing genes from Thraustochytrid microalgae using a plasmid vector which can replicate itself without being inserted into chromosomal DNA in the Thraustochytrid microalgae having producing ability of bio-oil containing high concentration of polyunsaturated fatty acids such as DHA, etc. The method can be utilized for the efficient expression of a variety of genes in Thraustochytrid microalgae.

Description

{Novel method for gene expression in Thraustochytrid microalgae}

The present invention relates to a novel method for gene expression in trout kits lead microalgae.

Thraustochytrid, an organic heterotrophic microalga that occupies the lowest rank in the marine ecosystemic food chain, contains triacylglycerol, which contains a high concentration of various polyunsaturated fatty acids including DHA (docosahexaenoic acid) As a source of supply. Polyunsaturated Fatty Acids DHA is a fatty acid essential for the brain, eye tissues and nervous system, and is known to play an important role in the development of visual and motor neurological skills, especially in infants. In addition, it has been reported that the amount of dementia in the brain of the dementia is remarkably decreased, and various anti-aging functions such as suppression of the macular degeneration of the presbyter are newly discovered. Most high-end animals, including humans, must ingest polyunsaturated fatty acids as essential nutrients because they can not synthesize the polyunsaturated fatty acids required for normal biological functioning themselves, and the World Health Organization recommends that DHA-containing polyunsaturated fatty acids Is recommended to continue to eat. Traditionally, the sources of DHA polyunsaturated fatty acids are deep sea fish, which make up the top of marine ecosystems such as tuna and salmon. However, as the pollution of the marine environment becomes worse, the risk of ingestion of deep sea fish is increasing due to the accumulation of pollutants such as mercury, heavy metals, environmental hormones and radioactive substances in the body of deep sea fish. Therefore, as a new means to safely and reliably supply DHA polyunsaturated fatty acid oil, Trout kettled microalgae is of great industrial value.

Various methods have been suggested for gene overexpression in trout-to-kit lead microalgae. Since the first introduction of the genetic transformation method of the microorganisms of Troutoquitrid using acetolactate synthase as a selectable marker by Marquette, Troutokitrid microalgae using various antibiotic resistance genes as selection markers Transformation techniques have been reported. However, the genetic transformation technology developed in the microorganisms of the present invention has the advantage that the introduced gene is stably maintained by the chromosomal integration of the introduced gene into the chromosomal DNA, The expression of the gene is limited in comparison with the gene expression method using the centromeric or episomal plasmid.

Accordingly, the present invention has developed a gene expression technique using an autonomously replicating plasmid in the troutocide reductase microalgae, which can be effectively applied for the expression of various genes.

Korean Patent Laid-Open Publication No. 2015-0084148 discloses a recombinant vector and its use for increasing biomass and lipid productivity of microalgae, and Korean Patent No. 0443843 discloses a method of using microalgae Biosynthesis of exogenous proteins' has been disclosed. However, as described in the present invention, there is no known new method for gene expression in microorganisms of Trout kettle.

The present invention provides a method for overexpressing a gene in a microorganism of Trout kettle using a plasmid vector that can be replicated by itself without being inserted into a chromosomal DNA in a microalgae of Trout kettled line And that the content of polyunsaturated fatty acids in the microalgae transformed with the vector of the present invention is higher than that of the non-transformant, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention provides a CEN / ARS replication starting point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, Wherein the target gene is overexpressed in a trout kittite microalgae comprising the steps of:

The present invention also relates to a method for detecting a CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, The method comprising the steps of: (a) cultivating a transformed strain of Troutkit lead microalgae.

In addition, the present invention provides transformed trout kittill microalgae produced by the above method.

The present invention also relates to a method for detecting a CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a target gene expression cassette comprising a promoter of a gene derived from a Trotsky kit lead, a multiple cloning site (MCS) for insertion of a target gene and a transcription termination factor of a gene derived from a Trotsky kit lead, The expression vector provides an autonomously replicating recombinant vector.

The present invention relates to a method for overexpressing a gene in a microorganism of a Troutukitrid using a plasmid vector capable of replication by itself without being inserted into a chromosomal DNA in a microorganism of a Troutukitrid line, Can be utilized for efficient expression of genes.

1 shows a process for producing the plasmid vector p415-Hyg of the present invention.
FIG. 2 shows the result of analysis of the plasmid p415-Hyg of KRS101 transformant of Aurantiochytrium sp. By restriction enzyme treatment (A) and antibiotic gene PCR amplification (B). 1: p415-Hyg plasmid DNA derived from KRS101 transformant strain of aurantiochitirit genus, and 2: p415-Hyg plasmid DNA derived from E. coli.
Fig. 3 shows a process for producing a plasmid for expression of delta-9 desaturase (9des) gene derived from Mortierella alpina .
FIG. 4 is a graph showing the expression of a delta-9 desaturase (9 des) gene expression plasmid (p415-Hyg-9des) of a KRS101 transformant strain of aurantoocytic genus in an antibiotic and a delta- PCR amplification. 1 to 3: p415-Hyg-9des plasmid DNA derived from aurantiochitirit KRS101 transformed strain, 2: p415-Hyg-9des plasmid DNA derived from Escherichia coli.
FIG. 5 shows a process for producing a plasmid for expressing the Thraustochytriidae sp. D1C-derived unsaturated enzyme SeqD1 gene.
FIG. 6 shows a process for producing a plasmid for expressing SeqD2 gene derived from Troutocid lead microtreating Trotsky tripe d1C-derived unsaturated enzyme.
FIG. 7 shows a process for producing a plasmid for expressing the elongase SeqE1 gene derived from Troutocide reed microalga Troutkottori large d1C.

In order to achieve the above object,

(1) CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, ;

(2) introducing the self-replicating recombinant vector into a microtide of trout to obtain a transformed microtide of Trout kettle; And

(3) culturing the transformed strains of the microorganisms of the present invention, and (3) culturing the transformed strains of the microorganisms.

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. In the present invention, plasmids and vectors are sometimes used interchangeably. The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

An " expression cassette "comprises three major components: i) a promoter; ii) a "coding polynucleotide," or "coding sequence," which is operably linked to the promoter and whose transcription is indicated by the promoter when the expression cassette is introduced into the cell, Quot; gene "); " polynucleotide " And iii) a terminator polynucleotide (also referred to as a transcription termination factor) that directs termination of transcription and is located immediately downstream of the second polynucleotide.

In the recombinant vector of the present invention, the promoter in the expression cassette and the transcription termination factor are preferably selected from the group consisting of a housekeeping gene derived from a trotsky kit lead, such as actin, ubiquitin, tubulin, , A heat shock protein 70, an elongation factor, and a superoxide dismutase (SOD) -coding gene, but the present invention is not limited thereto.

According to an embodiment of the present invention, step (1) of the method of overexpressing a target gene in the trout kitsch microalgae comprises the step of cloning CEN / ARS; A marker comprising a promoter of a KRS101-derived ubiquitin gene of a genus Aurantiochitium comprising the nucleotide sequence of SEQ ID NO: 1, a selection marker, and a transcription termination factor of a Uricitin gene derived from KRS101 of aurantoitium genus comprising the nucleotide sequence of SEQ ID NO: Expression cassettes; And a promoter of a KRS101-derived actin gene from aurantitium genus consisting of the nucleotide sequence of SEQ ID NO: 3, a target gene, and a transcription termination factor of a KRS101-derived actin gene from aurantoitium genus comprising a nucleotide sequence of SEQ ID NO: 4 A self-replicating vector for the expression of the desired gene can be produced in a trout kittlet microalga containing the target gene expression cassette.

The trout tochitrid microalgae may be selected from the group consisting of Aplanochytrium sp., Aurantiochytrium sp., Japonochytrium sp., Labyrinthula sp. But are not limited to, Oblongichytrium sp., Schizochytrium sp., Thraustochytrium sp., Or Ulkenia sp. .

"Target gene" means a gene encoding a protein to be expressed. The target gene is not particularly limited, and a polynucleotide encoding a desired protein may be appropriately selected and employed. The polynucleotide which is the target gene can be obtained by using a known technique such as PCR based on the nucleotide sequence information.

According to one embodiment of the present invention, the target gene is a gene encoding a delta-9 desaturase derived from Mortierella alpina , a gene encoding Thraustochytriidae sp. D1C A SeqD2 gene coding for an uncharacterized enzyme, a SeqD1 gene coding for an uncharacterized enzyme, a SeqD2 gene encoding a Thraustochytriidae sp. D1C-derived unsaturated enzyme, and SeqE1 encoding a Trotsky tripe d1C-derived elongase But it is not limited thereto.

In addition,

(1) CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, ; And

(2) introducing the self-replicating recombinant vector into a microorganism of a Troutukitrid to obtain a transformed microorganism of Troutukitrid; and .

The gene derived from the Trotsky kit lead is as described above.

According to one embodiment of the present invention, a method for producing transformed strachitlet microalgae is preferably (1) a CEN / ARS replication starting point; A marker comprising a promoter of a KRS101-derived ubiquitin gene of a genus Aurantiochitium comprising the nucleotide sequence of SEQ ID NO: 1, a selection marker, and a transcription termination factor of a Uricitin gene derived from KRS101 of aurantoitium genus comprising the nucleotide sequence of SEQ ID NO: Expression cassettes; And a promoter of a KRS101-derived actin gene from aurantitium genus consisting of the nucleotide sequence of SEQ ID NO: 3, a target gene, and a transcription termination factor of a KRS101-derived actin gene from aurantoitium genus comprising a nucleotide sequence of SEQ ID NO: 4 Preparing a self-replicating recombinant vector for expression of a target gene in a trout kittlet microalga containing a target gene expression cassette; And

(2) introducing the self-replicating recombinant vector into the microtidal strains to obtain transformed strains of microorganisms of the genus Trastuzukitrid, and .

In addition, the present invention provides transformed trout kittill microalgae produced by the above method.

According to one embodiment of the present invention, the transformed strains of the microorganisms are characterized by having a higher polyunsaturated fatty acid content than non-transformants. The polyunsaturated fatty acids may be selected from the group consisting of linoleic acid, arachidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid, DHA ), And adrenic acid, but the present invention is not limited thereto.

The present invention also relates to a method for detecting a CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a target gene expression cassette comprising a promoter of a gene derived from a Trotsky kit lead, a multiple cloning site (MCS) for insertion of a target gene and a transcription termination factor of a gene derived from a Trotsky kit lead, The expression vector provides an autonomously replicating recombinant vector.

The recombinant vector is preferably a CEN / ARS replication origin; A marker comprising a promoter of a KRS101-derived ubiquitin gene of a genus Aurantiochitium comprising the nucleotide sequence of SEQ ID NO: 1, a selection marker, and a transcription termination factor of a Uricitin gene derived from KRS101 of aurantoitium genus comprising the nucleotide sequence of SEQ ID NO: Expression cassettes; And a nucleotide sequence of SEQ ID NO: 3, a MCS (multiple cloning site) for insertion of a target gene, and a nucleotide sequence of a nucleotide sequence of SEQ ID NO: 4, a promoter of a KRS101-derived actin gene from KRS101 in aurantiochitium genus But is not limited to, a transcriptional recombinant vector for expression of a desired gene in a trout kittlet microalga containing a target gene expression cassette containing a transcription termination factor of the gene.

The recombinant vector is inserted into a target gene expression cassette in which a target gene whose expression is regulated by a promoter of KRS101-derived actin gene derived from aurantititium genus belonging to the nucleotide sequence of SEQ ID NO: 3 is inserted, and MCS is inserted into the cassette by a method known to a person skilled in the art Can be suitably produced.

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 merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example 1. Construction of self-replicating plasmid vector of trout kits lead microalgae using yeast CEN / ARS

A plasmid vector capable of independent replication without being inserted into the chromosomal DNA was constructed as shown in Fig. 1 in the trout squirt lead microalgae. Hygromycin (Hyg) resistance gene (XP_003071652.1) was inserted into the promoter (SEQ ID NO: 1) of KRS101-derived ubiquitin gene from the microorganism aurantiochytrium sp. Aurantiochitrium sp. And the transcription termination sequence No. 2), and then inserted into a yeast CEN / ARS plasmid to prepare a p415-Hyg plasmid.

Example 2. Fabrication of transformant strains of microorganism strains with yeast CEN / ARS plasmid vector

The p415-Hyg plasmid isolated from Escherichia coli host was introduced into KRAS101 strain of aurantiocytrium, which is a micro-algae of trout kits, through electrophoresis to obtain a medium containing glucose (50 mg / ml) containing hygromycin The strains showing resistance to g / L, nitrogen source Yeast extract 10 g / L, artificial sea salt 5 g / L, and KH ₂ PO ₄ 9 g / L were isolated and then transformed into a stable strain Were selected. The presence of p415-Hyg was confirmed by restriction enzyme treatment and antibiotic gene amplification using the plasmid DNA isolated from the obtained transformant (Fig. 2).

Example 3. Overexpression of delta-9 desaturase using self-replicating plasmid vector

The delta-9 desaturase gene (AB015611.1) derived from Mortierella alpina was overexpressed in a trout-kittide microalga using the self-replicating plasmid vector. As shown in FIG. 3, the M. alpina delta-9-unsaturated enzyme gene was inserted into the promoter (SEQ ID NO: 3) of KRS101-derived actin gene (SEQ ID NO: 3) of a strain of trout kitside microalga aurantii kitsium and a transcription termination sequence No. 4), and p415-Hyg-9des inserted into the p415-Hyg plasmid vector were prepared. Plasmid DNA prepared in KRS101 strain of aurantiocytrium, which is a microorganism of Trouschitit reid, was introduced into a medium containing hygromycin (1 mg / ml) (carbon source glucose 50 g / L, nitrogen source The strains showing resistance to yeast extract 10 g / L, artificial sea salt 5 g / L and KH ₂ PO ₄ 9 g / L were isolated and stable transformants were selected by subculture 4). The selected transformant strain was cultured at 28 ° C and 120 rpm for 3 days using the medium described above. The cells were recovered, lyophilized, resuspended in 5% methanolic sulfuric acid solution and incubated at 90 ° C for 1 hour The fatty acid ester produced by the reaction was extracted with nucleic acid and analyzed by gas chromatography.

As a result, as shown in Table 1, the content of oleic acid (C18: 1) and linoleic acid (C18: 2) in the transformant strain into which p415-Hyg-9des plasmid was introduced was significantly , Indicating a clear overexpression of the delta-9-unsaturated enzyme gene.

On the other hand, a transformant strain in which a delta-9-desaturase gene was introduced by chromosomal integration was prepared and the fatty acid composition was analyzed. As a result, the p415-Hyg-9des plasmid of the present invention was introduced (Wild strains), unlike the transformed strains of the present invention.

Example 4: Preparation of microsaturated fatty acid enhancing strains of microtidal transformants

Using the plasmids vector, a transformant strain overexpressing the unsaturated and extracellular genes derived from microtusculus strains was prepared. As shown in FIG. 5, an expression plasmid vector (SeqD1) (SEQ ID NO: 5) derived from a Troutokitrid microalga Trostokitri larva d1C (KCTC 12769BP) was prepared (p415-Hyg-SeqD1 ), And a KRS101 transformant strain of aurantiochitium genus introduced thereinto was selected. As shown in Table 2, the content of palmitic acid (C16: 0) was greatly decreased and the content of arachidonic acid (C20 : 4ω6), eicosapentaenoic acid (EPA) (C20: 5ω3), docosapentaenoic acid (C22: 5ω6, C22: 5ω3), docosahexaenoic acid, DHA ) (C22: 6? 3) were found to increase in all the polyunsaturated fatty acid contents.

As shown in FIG. 6, another plasmid vector (SeqD2) (p415-Hyg-SeqD2) was produced by introducing another unsaturated enzyme gene (SeqD2) (SEQ ID NO: 6) derived from Troutokitri large- The transformed KRS101 strain transformed into aurantiocytrium was introduced. As shown in Table 3, the content of palmitic acid (C16: 0) was greatly decreased and the content of docosapentaenoic acid (C22: 5? 6, C22: (C20: 4ω6) and EPA (C20: 5ω3) were found to be decreased compared with the control, while all polyunsaturated fatty acid contents of DHA (5ω3) and DHA (C22: 6ω3)

(P415-Hyg-SeqE1) expression vector (SeqE1) (SeqE1) derived from Trochutriot larvae d1C, which is a microorganism of trophic kits, as shown in FIG. 7, One KRS101 transformant strain of aurantiochitirit was selected. The selected transformant strains were cultured as described above and the fatty acid composition was analyzed. As shown in Table 4, the content of palmitic acid (C16: 0) was decreased while the content of docosapentaenoic acid (C22: (C22: 6? 3) was significantly higher than that of the control, while adrenic acid (C22: 4? 6) content was significantly increased.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Novel method for gene expression in Thraustochytrid microalgae <130> PN16322 <160> 7 <170> Kopatentin 2.0 <210> 1 <211> 997 <212> DNA <213> Aurantiochytrium sp. KRS101 <400> 1 gaatacctta aggagagttt ggagagttat cctggattaa ggtgtattcg aaactgtgcg 60 cctggccaat ttccttaacg tggccgggga tttattacac agttactccg agtcgtggac 120 taggctttca tgaccctgtg tgaggtttac ccgagtcgag agggtgtgcc ttcaacactg 180 tggaagaact gccagtgggg atgtgagagg gaaattcttc agtggggcat agttggaagc 240 ggtctcgaga gcggccgcgg gagactttga aaggcacagt aagtttttga aagataaaag 300 ggataaagat caaaaaggtc atcctggcag acatgtggaa atccaagagc actgaaaacg 360 cgaaggttct caggtctctg ctccatgttc atggcagtcc gaacggcttt gcctaagact 420 cggtactatg ctacagctcc agaagtcagt cagtcagtct gtcagtctga gcaaaatgag 480 cgatttttta aatcctttgc gaagcttttt cgtttgaaag ttgtttcttt aaaatggaaa 540 gttgattgat tgaccccgat cactgaagct ttatcgcaag aggcagagag aacagacggt 600 gcgagatggg tattattgta aacgctatgc gcgcaatagc atccaatcaa gagcgcaatc 660 gagcaacaga cggagatcaa aaaataagat aataaaaata aaactgacta tatgtaggag 720 tggttatgga ttttgctaag atactggcga ttgtgttgaa ttgaaaagaa agaaatagaa 780 gaagaagaag aagagagaac aatgctttta ttttacaaag cagcagctta ctttaaagtg 840 cttttcatgg actgagcaga atcactcgag gcgcagcaaa aacccaactc cagccaggcc 900 agtccagacc gaagaaagca agcgtaggga gctcagcttg atcctggtgc tgttcattga 960 gattgacaaa gttgtaagaa ttagataagc aaacaac 997 <210> 2 <211> 693 <212> DNA <213> Aurantiochytrium sp. KRS101 <400> 2 ttatatacct acctctttcc acataaacaa actctttacg cgctgttttg attttccatc 60 tttgacctaa gagatcactc tctcctatct tctcgtactt cgtcattgtt cgcacctcta 120 tgcagcaagt ctcagctatt agatgtgctg ggccggccgt gtatgtgcac gcttccatct 180 ttatttcagc cttgggcatc atctcatttg tgctgctccc catcctccct aaagttcctc 240 aacgttagtc atcatgcctt cgcacactag gcatttttat gaattgaata gtatctctat 300 ctggagaaat ctaatctgaa tattctgctc aaaatcaccc cctatatttt tgacactcat 360 tcttgaactt gaagttatca gtgttttatt cagctgtaga tcaaccgagc gataatatct 420 agatccaaaa cattacgaat tttgcaccaa cctttcaaag cttctgcacg ttttaatatc 480 accacataat gatgctcaga tggcaaaagc caccgtgacg agtatgttgt gccatctgag 540 aagacaatga tattatgata ctaccatctc catccttacc acagcatcat gacatccaat 600 accttgggac cacaaagcta gtaatggagg catgtttaac ttgccggtgt agatactgta 660 ccatgtagtt ttcttatgca cgaaaagaga aga 693 <210> 3 <211> 698 <212> DNA <213> Aurantiochytrium sp. KRS101 <400> 3 gtagagccca gcaaagtttt aattaaaagc taaagtatat atagcatatt gaaaattatt 60 cttttgtaaa gctaaaaatt aaaagtataa tagatgccct atattaaaca atttttatcg 120 aactaagaaa acagaagagt aggtagcgaa aattggaact ggggtggcaa gagagttcac 180 actttctttt cgtaagttct tttggttgag gaagttagtt agttagttag ttatttgttt 240 agttgtgcta tccgtatgtt tccatttgac tgtctgtgta tctatctatc tgtttgactc 300 actcactcat cttttcacaa ttctcgcaag tgaggggggg gcatctcgac tttctcgcga 360 ttttcttcaa gatccccctc ccgccgcact ggggtgcttt actgaggcaa aagctccagt 420 ttgagatgga aaggaagtgc agctaggggg aagaggggtg tgtttgtgag ggggaaatga 480 ggcagcagtc cgggtgcccc tcagaggcag tggcgatgag agggggtgtg agggggtgga 540 tttcgaaagg atcctcctta agtggaggca ttcgagagag ggtgcctgcc agctggcggt 600 atcgtggtcg cgacggctgc gctccaggat cagcaaaccc gcaacctcaa gctcaagaag 660 caacaacaca gtagcagaac aagcacccaa ctagcaaa 698 <210> 4 <211> 508 <212> DNA <213> Aurantiochytrium sp. KRS101 <400> 4 gttgctgaaa tcctggccta gagtaggagg gacttgtcga gttgtaggct tcagtgcttc 60 ctcgactcga gtccgagcct ctgcaggtga gattgtttca gtatagctta tgagcttgtg 120 ttgcttgttg tgctgcacat aacgtacata caaccataaa taaacacccc caaaaccctt 180 tgcccataat catgaattac ataaattcaa ctctgttaca gattcatcct cttttaccaa 240 caacttgtca aaacaatatc ctatcttgga attgaaattt atgaaccctt ctcgatggtt 300 agatattatt gctgaccttt aatttttgaa gttcgacttt gtatcgcgcc ttacgatatg 360 tctttgtcga tcgagttctg tgtgttctcc tacgttgatt ccttttggct cttcatggta 420 gtgtttttac agggaatgta gctttctttc agcatggatc cattcatatc ttttaccact 480 taaatatgca aatgggtgtc ttggtaga 508 <210> 5 <211> 1320 <212> DNA <213> Thraustochytriidae sp. d1C <400> 5 atgggcaagg gcagcgaggg ccgcagcgcg gcgcgcgaga tgacggccga ggcgaacggc 60 gacaagcgga aaacgattct gatcgagggc gtcctgtacg acgcgacgaa ctttaagcac 120 ccgggcggct cgatcatcaa cttcttgacc gagggcgagg ccggcgtgga cgcgacgcag 180 gcgtaccgcg agtttcatca gcggtccggc aaggccgaca agtacctcaa gtcgctgccg 240 aagctggatg cgtccaaggt ggagtcgcgg ttctcggcca aagagcaggc gcggcgcgac 300 gccatgacgc gcgactacgc ggcctttcgc gaggagctcg tcgccgaggg gtactttgac 360 ccgtcgatcc cgcacatgat ttaccgcgtc gtggagatcg tggcgctctt cgcgctctcg 420 ttctggctc tgtccaaggc ctcgcccacc tcgctcgtgc tgggcgtggt gatgaacggc 480 attgcgcagg gccgctgcgg ctgggtcatg cacgagatgg gccacgggtc gttcacgggc 540 gtcatctggc tcgacgaccg gatgtgcgag ttcttctacg gcgtcggctg cggcatgagc 600 gggcactact ggaagaacca gcacagcaag caccacgccg cgcccaaccg cctcgagcac 660 gatgtcgatc tcaacacgct gcccctggtc gcctttaacg agcgcgtcgt gcgcaaggtc 720 aagccgggat cgctgctggc gctctggctg cgcgtgcagg cgtacctctt tgcgcccgtc 780 tcgtgcctgc tcatcggcct tggctggacg ctctacctgc acccgcgcta catgctgcgc 840 accaagcggc acatggagtt cgtctggatc ttcgcgcgct acattggctg gttctcgctc 900 atgggcgctc tcggctactc gccgggcacc tcggtcggga tgtacctgtg ctcgttcggc 960 cccggctgca tttacatttt cctgcagttc gccgtcagcc acacgcacct gccggtgacc 1020 aacccggagg accagctgca ctggctcgag tacgcggccg accacacggt gaacattagc 1080 accaagtcct ggctcgtcac gtggtggatg tcgaacctga actttcagat cgagcaccac 1140 ctcttcccca cggcgccgca gttccgcttc aaggaaatca gtcctcgcgt cgaggccctc 1200 ttcaagcgcc acaacctccc gtactacgac ctgccctaca cgagcgcggt ctcgaccacc 1260 tttgccaatc tttattccgt cggccactcg gtcggcgccg acaccaagaa gcaggactga 1320                                                                         1320 <210> 6 <211> 1566 <212> DNA <213> Thraustochytriidae sp. d1C <400> 6 atgacggtcg ggtacgacga ggagatcccg tttgaaaagg tccgcgcgca caacaagccg 60 gacgacgcct ggtgcgcgat ccatggacac gtgtacgatg tgaccaagtt tgcgagcgtc 120 cacccgggcg gcgacatcat tttgctggcc gcgggcaaag aagccaccgt gctgtacgag 180 acttaccatg tgcgcggcgt ttcggatgcg gtgctgcgca agtatcgcat tggtaagctc 240 cccgacggcc agggaggcgc gaacgagaag gaaaaacgga cgctctcggg cctcacctcg 300 gcctcgtact acacgtggaa cagcgatttc tacaaggtga tgcgcgagcg ggtcgtggcg 360 cgcctcaagg agcggggcgt ggcgcggcgc ggcggctatg agctgtggac caaggcgttt 420 ctcctgctcg tgggcttttg gtcgtccttg tacctcatgt gcaccttgga cccgtccttt 480 gt; cagcacgatg gtaatcacgg cgcctttgcg cagacgcgat gggtgaacaa ggttgctggc 600 tggacgctgg acatgattgg cgccagtggc atgacgtggg agttccagca cgtgctcggg 660 caccacccgt acacgaacct gatcgaggag gacaatggtc tgcaaaaggt gagcggcaag 720 aagatggaca cacagacggc ggatcaggag agcgacccgg acgtgttttc gacgtacccg 780 atgatgcgtc ttcacccgtg gcatcaaaag cgctggtacc atcgtttcca gcacatttat 840 ggacccttta tctttggctt catgaccatc aacaaggtcg tcactcagga tgtgggcgtg 900 gtccttcgca agcgtctctt tcagatcgac gccgagtgca gatacgccag ccccttgtac 960 gtggctcgtt tctggatcat gaaagccctc accgtgctgt acatggtcgg gcttccctgc 1020 tatatgctcg gtccgtggca gggcctcaag ctctttgcta ttgcgcattt tacgtgcggc 1080 gaggtgctcg cgaccatgtt cattgtgaat cacatcattg agggcgtctc gtacgcctcg 1140 aaggacgcgg tgaagggaac gatggcgccc cccaagacgc tccacggtgt cacgccgatg 1200 ccgagacgc gcgaagaggc ctctgcccag gcgtcaaagg cgggcactgc ggtcaagtct 1260 gtcccgctcg acgactgggc cgccgtacag tgtcagactt ctgtgaattg gagtgtggga 1320 tcgtggttct ggaaccactt ttccggaggt ctcaatcacc aaattgagca tcatttgttc 1380 cctggcctga gccatcagac gtactatcac attcaggacg tcgtagagtc tacgtgtgcc 1440 gagtatggcg ttccgtatca gcacgaggcc tcgctgtggt ctgcgtactg gaagatgctt 1500 gagcatcttc gtcaacttgg caacgaggag gtgcatgagg cgtggcagca tgcgacgcgg 1560 gcttga 1566 <210> 7 <211> 834 <212> DNA <213> Thraustochytriidae sp. d1C <400> 7 atggacgtgg ccatggagca atggaagcgg ttcgtggaga cggtggacaa ggggattgta 60 gactttatgg aaggcgaaaa gaccaatgaa atgaatgccg gcaagccgct catctccacc 120 gaggagatga tggccctcat tgtgggctac ctcgcctttg ttgtttttgg ctcgggattc 180 ccacaacatt 240 ttccttactg ggctttccat gtacatggcc accgagtgcg cgcgccaggc gtaccttggc 300 ggctacaagc tatttggcaa ccccatggag aagggcaacg aggcgcacgc accgggtatg 360 gccaacatca tctacatttt ttacgtgagc aaatttctcg agttcctcga cactgtgttc 420 atgatcctcg gcaagaagtg gaagcagctc agcttcttgc acgtatacca tcacgcgagc 480 attagcttca tttggggtat catcgctcgt ttcgctcctg gcggagacgc atacttttcc 540 acgattttga acagctgcgt ccatgtcatg ctctacggct actacgcgtc caccacgctc 600 ggctacggct tcatgcgccc tcttcgtccg tacattacca cgattcagct cacacagttt 660 atggccatgg tcgtccagtc cgtctatgac ttttacaacc cttgcgacta cccgcagcct 720 ctcgtcaagc ttctcttttg gtacatgctt accatgctcg gcctctttgg caacttcttc 780 gtgcagcagt acctgaagcc caagccggcc accaagaagc aaaagaccat ctaa 834

Claims (11)

(1) CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, ;
(2) introducing the self-replicating recombinant vector into a microtide of trout to obtain a transformed microtide of Trout kettle; And
(3) culturing the transformed Trout k Kitt microalgae, wherein the target gene is overexpressed in a microorganism of Trout kettle. The method according to claim 1, wherein the Troutukitrid gene is selected from the group consisting of actin, ubiquitin, tubulin, heat shock protein 70, elongation factor and superoxide dismutase dismutase) coding gene, wherein the target gene is overexpressed in a microorganism of the present invention. The microorganism of claim 1, wherein the microorganism strains are selected from the group consisting of Aplanochytrium sp., Aurantiochytrium sp., Japonochytrium sp. It is also possible to use a compound of the formula ( I ) or a salt thereof , such as Labyrinthula sp., Oblongichytrium sp., Schizochytrium sp., Thraustochytrium sp. Or Ulkenia sp. A method for overexpressing a gene of interest in a microtidal strain of Trousky kits. The method according to claim 1, wherein the target gene is selected from the group consisting of a gene encoding delta-9 desaturase derived from Mortierella alpina , a gene encoding Thraustochytriidae sp. D1C-derived unsaturated enzyme A SeqD2 gene coding for a Thraustochytriidae sp. D1C-derived unsaturated enzyme, and a SeqE1 coding for an elongase derived from a Trotsky tripe. Wherein the target gene is overexpressed in a trout kitteed microalgae. (1) CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a gene expression cassette containing a transcription termination factor of a gene derived from a Troutukitrid gene, a target gene and a transcription termination factor of a gene derived from a Troutukitrid, ; And
(2) introducing the self-replicating recombinant vector into a microtidal strain of Troutukitrid to obtain transformed microorganism of Troutukitrid microalgae. The microorganism of claim 5, wherein the microorganism strains are selected from the group consisting of Aplanochytrium sp., Aurantiochytrium sp., Japonochytrium sp. It is also possible to use a compound of the formula ( I ) or a salt thereof , such as Labyrinthula sp., Oblongichytrium sp., Schizochytrium sp., Thraustochytrium sp. Or Ulkenia sp. Wherein the transformed strains are cultured in the presence of a microorganism. 6. The method according to claim 5, wherein the target gene is selected from the group consisting of a gene encoding delta-9 desaturase derived from Mortierella alpina , a gene encoding Thraustochytriidae sp. D1C-derived unsaturated enzyme A SeqD2 gene coding for a Thraustochytriidae sp. D1C-derived unsaturated enzyme, and a SeqE1 coding for an elongase derived from a Trotsky tripe. &Lt; / RTI &gt; wherein the transformed strains are selected from the group consisting of: A transformed Trout kittill microalgae produced by the method of any one of claims 5 to 7. [Claim 9] The transformed straitukitrid microalgae according to claim 8, wherein the transformed strains have increased polyunsaturated fatty acid content compared to the non-transformant. 10. The composition of claim 9, wherein the polyunsaturated fatty acid is selected from the group consisting of linoleic acid, arachidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid, Wherein the transformed strains are at least one selected from the group consisting of docosahexaenoic acid (DHA) and adrenic acid. CEN / ARS replication start point; A marker expression cassette comprising a promoter of a gene derived from Troutukitreid, a selection marker coding gene and a transcription termination factor of a gene derived from Troutukitrid; And a target gene expression cassette comprising a promoter of a gene derived from a Trotsky kit lead, a multiple cloning site (MCS) for insertion of a target gene and a transcription termination factor of a gene derived from a Trotsky kit lead, Expression-replicating recombinant vector.

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