CN102492700A - DNA sequence for coding delta15 fatty acid desaturase (FAD) of Myrmecia incisa and application thereof - Google Patents

Abstract

The present invention relates to a DNA sequence encoding the Δ15 fatty acid desaturase of Chlorella nigrum, said DNA sequence comprising: (a) the nucleotide sequence described in SEQ ID NO.10, or (b) and (a) The nucleotide sequence complementary to the nucleotide sequence. The present invention also provides a recombinant expression vector containing the above nucleotide sequence, a genetically engineered host cell and its application. The present invention has the advantages of cloning the fatty acid desaturase gene of green algae with a nicked edge and identifying its function, laying the foundation for elucidating the synthesis and metabolism pathway of long-chain polyunsaturated fatty acids in green algae with a nicked edge In vivo genetic manipulation to achieve efficient synthesis of ω3 long-chain polyunsaturated fatty acids creates conditions.

Description

A DNA sequence encoding Δ15 fatty acid desaturase of green algae with nick margin and its application

technical field

The present invention relates to a DNA sequence, specifically, a DNA sequence encoding the Δ15 fatty acid desaturase of green algae nicked margin and its application.

Background technique

ω3 long-chain polyunsaturated fatty acid (ω3 polyunsaturated fatty acid, ω3 PUFA) mainly includes α-linolenic acid (C18:3 ^{Δ9, 12, 15} , ALA), eicosapentenoic acid (eicosapentenoic acid, C20:5 ^{Δ5, 8, 11, 14, 17} , EPA) and docosahexenoic acid (C20:6 ^{Δ4, 7, 11, 13, 16, 19} , DHA). It has a variety of physiological and pharmacological activities in the human body, and can prevent cardiovascular and cerebrovascular diseases and inhibit tumor growth. For example, DHA and EPA participate in the lecithin of the neuronal cell membrane, which improves the liquid fluidity and permeability of the cell membrane; DHA plays an important role in the division, proliferation, nerve conduction, synaptic growth and development of brain nerve cells. It plays an important role and is an essential substance for brain synthesis and IQ development, especially for the baby's vision and neurodevelopment. ω3 PUFA can also reduce the content of arachidonic acid (arachidonic acid, C20:4 ^{Δ5, 8, 11, 14} , ArA) in the phospholipids of multinucleated leukocytes and mast cells, and reduce its release when allergic reactions occur, thereby reducing white three Oxygen production, reduces inflammation.

Although ω3 PUFA plays an important role in human health, ω3 PUFA is an essential fatty acid for humans, that is, the human body cannot synthesize ω3 PUFA itself, but must be obtained from food.

Due to the high content of ω3 PUFA in seafood, the current commercial sources of ω3 PUFA are mainly seafood, such as fish oil on the market. However, fish oil has the disadvantages of heavy fishy smell, poor taste, cholesterol and erucic acid, and the lack of marine fish resources. Therefore, it is urgent for researchers in nutrition, hygiene, biology and other fields to find suitable ω3 PUFA raw materials. unresolved issues. Among them, ALA in plants can be converted into EPA and DHA under the catalysis of suitable enzymes, which also has similar functions to fish oil, and is stable in nature, easy to obtain, and free of cholesterol and erucic acid. Therefore, the recent research on plant ALA is particularly important. Attention and concern. Myrmecia incisa is a single-celled green alga belonging to the phylum Chlorophyta and the class Trebouxiophyceae. Cells often gather together to form cell clusters similar to amorphous groups. Studies have found that the algae contains ω3 PUFA such as ALA and EPA. Compared with other microalgae, the algae has a higher PUFA content, which is conducive to the industrial production of PUFA through microalgae bioreactors and the realization of high-value microalgae products.

In the PUFA synthesis pathway of microalgae, microalgal fatty acid desaturase (fatty acid desaturase, FAD) is the key enzyme for PUFA synthesis, and ω3 fatty acid desaturase (ω3 fatty acid desaturase, ω3 FAD) is the One of a kind in FAD. In microalgae, ω3 FAD mainly includes Δ15 FAD, Δ17 FAD and so on. Among them, Δ15 FAD can catalyze the desaturation of linoleic acid (linoleic acid, C18:2 ^{Δ9, 12} , LA) into ALA at the Δ15 position of green algae, and Δ17 FAD can catalyze ArA of green algae to produce EPA, thereby entering ω3 PUFA metabolic pathways. Therefore, the cloning and identification of the key enzymes for PUFA synthesis in microalgae—ω3 FAD is of great significance for the large-scale production of ω3 PUFA through genetic engineering.

Chinese patent document CN 200710300675.8, announced on September 7, 2011, discloses a marine microalgae Δ5 fatty acid desaturase, its encoding gene and its application. The marine microalgae Δ5 fatty acid desaturase provided by the invention can effectively Catalyze ETA (20:4 ^Δ8,11,14,17 ) to generate EPA (20:5 ^{Δ5,8,11,14,17} ); Chinese patent document CN 200810115245.3, announced on December 22, 2010, discloses a A marine microalgae Δ6 fatty acid desaturase, its coding gene and its application, the invention provides a marine microalgae Δ6 fatty acid desaturase that can effectively catalyze LA to generate SDA (18:4 ^Δ6,9,12,15 ). However, there is no report about the DNA sequence encoding Δ15 FAD of Chlorella nichei and its application.

Contents of the invention

The object of the present invention is to provide a DNA sequence encoding the Δ15 fatty acid desaturase of green algae Δ15 to address the deficiencies in the prior art.

Another object of the present invention is to provide a recombinant expression vector.

Another object of the present invention is to provide a genetically engineered host cell.

Another object of the present invention is to provide a use of the above-mentioned DNA sequence, recombinant expression vector and host cell.

For realizing above-mentioned object, the technical scheme that the present invention takes is:

Described DNA sequence comprises:

(a) the nucleotide sequence set forth in SEQ ID NO.10, or

(b) A nucleotide sequence complementary to the nucleotide sequence described in (a).

For realizing above-mentioned second purpose, the technical scheme that the present invention takes is:

The vector is a recombinant expression vector constructed by the above nucleotide sequence and plasmid or virus.

The plasmid in question is the pYES2 plasmid.

For realizing above-mentioned 3rd purpose, the technical scheme that the present invention takes is:

The host cell is selected from one of the following host cells:

(a) host cells transformed or transduced with the above nucleotide sequences and their progeny cells;

(b) Host cells transformed or transduced with the above-mentioned recombinant expression vectors and their progeny cells.

The host cells are bacterial cells, fungal cells, plant cells or animal cells, or progeny of these host cells.

The host cell is a yeast cell.

For realizing above-mentioned 4th object, the technical scheme that the present invention takes is:

The application of the DNA sequence, the recombinant expression vector or the host cell in the production of long-chain polyunsaturated fatty acids.

The long-chain polyunsaturated fatty acid is ω3 long-chain polyunsaturated fatty acid.

The long-chain polyunsaturated fatty acid is α-linolenic acid.

The present invention has the advantage that:

1. The present invention has cloned the ω3 FAD gene of Chlorella nicotii and carried out functional identification on it, confirming that it can encode Δ15 FAD enzyme, which can catalyze LA into ALA.

2. The present invention lays the foundation for elucidating the PUFA synthesis and metabolism pathways of Chlorella notch margin.

3. The present invention provides a theoretical basis for improving the ability of oil crops to synthesize PUFAs, and creates conditions for the efficient synthesis of ω3 PUFAs by genetic manipulation in organisms. If the gene is transferred to oil crops, it can effectively Desaturation of LA to ALA endows these oil crops with the ability to synthesize ω3 PUFAs to improve their quality.

Description of drawings

Accompanying drawing 1 is the electrophoresis pattern of 5'- and 3'-terminal RACE amplification and specific target bands.

Accompanying drawing 2 is the schematic diagram of the full-length structure of the ω3 FAD gene of Chlorella notch margin.

Accompanying drawing 3 is the detection result of the yeast fatty acid component without adding substrate group.

Figure 4 shows the detection results of the fatty acid components of yeast in the group added with the substrate LA (C18:2 ^{Δ9, 12} ).

Accompanying drawing 5 is the GC-MS spectrum of yeast fatty acid composition.

Detailed ways

The specific embodiments provided by the present invention will be described in detail below in conjunction with the accompanying drawings.

Example

1. Materials

(1) Myrmecia incisa Reisigl H4301 was obtained from the Culture collection of algae of Charles University of Prague. The culture medium was BG-11 under the condition of temperature of 25°C, light intensity of 115 μmol photons m ^-2 s ^-1 , light/dark ratio of 14 h/10 h.

(2) The pYES2 plasmid vector and the yeast INVSc1 strain (His ^– , Leu ^– , Trp ^– and Ura ^– deficient) were purchased from Invitrogen. Yeast was cultured in YPD medium at 28°C with shaking at 200 rpm.

2. Method

(1) Take 100 mg of fresh algae cells and place them in a pre-cooled mortar, add liquid nitrogen and grind them thoroughly.

(2) Genomic DNA was extracted by CTAB method and total RNA was extracted by Trizol method, and stored at -20°C for later use. Take 1-5 μg of RNA, and use a reverse transcription kit (TaKaRa Company) to synthesize the first strand of cDNA as a PCR reaction template for gene cloning.

2.5 μL，dNTP（各10 mM）2 μL，引物各1.0 μL（10 μM），模板1.0 μL，Taq酶0.5 μL（TaKaRa公司），无RNase水17 μL。PCR反应程序为：94℃预变性2 min，然后30个循环包括94℃变性30 s，58℃退火30 s，72℃延伸1 min，最后72℃延伸10 min。将PCR产物胶回收纯化后，与pMD19T载体（Promega公司）连接后转入大肠杆菌DH5α感受态细胞，筛选阳性克隆，菌落PCR验证后，将阳性克隆菌液送至上海桑尼生物科技有限公司测序得到缺刻缘绿藻ω3 FAD基因片段的序列。 (3) According to the conserved regions TMFWALF and HHDIGTH of the amino acid sequences of Chlamydomonas reinhardtii and Chlorella vulgaris ω3 FAD (GenBank accession numbers are ABL09485 and BAB78717, respectively), degenerate primer sequences were designed: upstream primer (SEQ ID NO.1): 5 '-ACCATGTTCTGGGC(C/T)CT(G/C)TT-3', downstream primer (SEQ ID NO.2): 5'-TG(G/C)GTGCC(A/G)ATGTCGTGGTG-3' (in In the attached sequence list, y represents C/T, s represents G/C, r represents A/G), which is used for the cloning of the cDNA fragment of the ω3 FAD gene of Chlorella nickers. PCR amplification was then performed using the following reaction system and procedures. 25 μL reaction system contains: 10×PCR buffer

2.5 μL, 2 μL of dNTP (10 mM each), 1.0 μL of each primer (10 μM), 1.0 μL of template, 0.5 μL of Taq enzyme (TaKaRa Company), 17 μL of RNase-free water. The PCR reaction program was: pre-denaturation at 94°C for 2 min, followed by 30 cycles including denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, and finally extension at 72°C for 10 min. After recovering and purifying the gel of the PCR product, it was connected with the pMD19T vector (Promega Company) and then transferred into E. coli DH5α competent cells to screen positive clones. After colony PCR verification, the positive clones were sent to Shanghai Sunny Biotechnology Co., Ltd. for sequencing Obtain the sequence of the ω3 FAD gene fragment of Chlorella notchi.

(4) Design gene-specific primers based on the obtained cDNA fragment sequence of the ω3 FAD gene of Chlorella nickers: GSP 1 (SEQ ID NO.3): 5'-ATGTGTGCGGTGGCTGATCCTCCAGC-3',

GSP 2 (SEQ ID NO. 4): 5'-CTTCTCCAGCAACAAGACGCTCAACAAC-3'. The 5′-RACE and 3′-RACE reverse transcription systems were respectively established according to the instructions of the SMART ^TM RACE cDNA Amplification Kit (Clontech Company), and then amplified according to the principle of touchdown PCR. 25 μL of 5′-RACE reaction system includes: 17 μL of PCR grade water, 2.5 μL of 10×Advantage 2 PCR buffer, 0.5 μL of dNTPs, 0.5 μL of GSP1, 2.5 μL of 10×UPM, 1.5 μL of 5′ - The RACE template is the first strand of cDNA obtained in step (2) and 0.5 μL of 50×Advantage 2 polymerase mixture. The 3′-end reaction system is the same as the 5′-end reaction system except for 0.5 μL of GSP 2 and 1.5 μL of 3′-RACE cDNA. The reaction program was: denaturation at 94°C for 30 s, extension at 72°C for 150 s, 5 cycles; denaturation at 94°C for 30 s, annealing at 70°C for 30 s, extension at 72°C for 150 s, 5 cycles; denaturation at 94°C for 30 s, 68°C Anneal for 30 s, extend at 72°C for 150 s, 30 cycles; extend at 72°C for 7 min. Then electrophoresis detection, gel recovery and purification, TA cloning and sequencing, and sequence splicing were performed to obtain the full-length cDNA sequence of the ω3 FAD gene of Chlorella japonicum.

(5) According to the full-length cDNA sequence of the ω3 FAD gene of Chlorella nickifolia obtained in step (4), design primers at the 5′-UTR and 3′-UTR of the gene: upstream primer (SEQ ID NO.5): 5'-AAACCCGGCAACAATGCA-3', downstream primer (SEQ ID NO.6): 5'-AGAATCAGAACCCGAAGC-3', clone the full-length DNA sequence of ω3 FAD gene. 25 μL of PCR amplification reaction system contains 2.5 μL of 10×PCR buffer, 2 μL of dNTP (10 mM each), 1.5 μL of Mg ²⁺ (25 mM), 1.0 μL of primers (10 μM), 1.0 μL template, 0.5 μL Taq enzyme (TaKaRa company) and 15.5 μL RNase-free water. The PCR reaction program was: pre-denaturation at 94°C for 5 min; then 35 cycles including denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 2 min; finally, extension at 72°C for 10 min. After the above PCR products were recovered and purified, they were all cloned by TA, and the positive clones were selected and sent to Shanghai Sunny Biological Co., Ltd. for sequencing to obtain the full-length DNA sequence of the ω3 FAD gene of Chlorella nickedlis.

(6) Design primers according to the pYES2 vector (Promega Company) and the ORF sequence of the ω3 FAD gene of Chlorella nickers: upstream primer (SEQ ID NO.7): 5'-cg GAATTC ATGCAGGCCCCCACTATGT-3', downstream primer (SEQ ID NO. .8): 5'-gc TCTAGA CTACACATCCGAGCCACTG-3', wherein, italic GAATTC and italic TCTAGA represent the restriction sites of Eco RI and Xba I, respectively. PCR amplification was carried out using the ω3 FAD cDNA of Chlorella nicklis as a template. The reaction system is as follows: 2.5 μL of 10×PCR buffer, 2 μL of dNTPs, 1.5 μL of Mg ²⁺ (25 mM), 1.0 μL of template, 1 μL of upstream and downstream primers, 16 μL of RNase-free water, 0.25 μL of Taq enzyme (5U/μL). The PCR reaction conditions were: pre-denaturation at 95°C for 5 min; 36 cycles including denaturation at 94°C for 1 min and annealing at 66°C for 1 min; extension at 72°C for 2 min, and finally extension at 72°C for 7 min. After the PCR products were recovered and purified for TA cloning, the positive clones were sent to Shanghai Sunny Biological Co., Ltd. for sequencing to ensure the accuracy of the sequence.

和XbaI进行双酶切消化反应，同时对pYES2质粒也进行EcoR / XbaI双酶切反应。反应体系为：4 μL的10×M缓冲液，4 μL的0.1% BSA，DNA≤2 μg，EcoR

及XbaI各1 μL，加无RNase水至20 μL。37℃消化反应4 h。割胶回收酶切后的目的片段，并用T₄ DNA连接酶将酶切后的ω3 FAD基因片段与pYES2片段连接得到重组载体pY-ω3 FAD。连接反应体系为：2.5 μL的缓冲液，ω3 FAD基因DNA片段约0.3 pmol，载体pYES2的DNA消化片段约0.03 pmol，1 μL的T₄ DNA连接酶，加无RNase水至25 μL。16℃连接过夜。连接后转化大肠杆菌DH5α感受态细胞，按照上述方法进行克隆、菌落PCR验证和测序。从大肠杆菌DH5α中抽提pY-ω3 FAD质粒，-20℃保存备用。 (7) Extract the pMD19T-ω3 FAD plasmid from Escherichia coli DH5α, and use the restriction endonuclease Eco R

Perform double enzyme digestion with Xba I, and Eco R for pYES2 plasmid / Xba I double enzyme digestion reaction. The reaction system is: 4 μL of 10×M buffer, 4 μL of 0.1% BSA, DNA≤2 μg, Eco R

and Xba I each 1 μL, add RNase-free water to 20 μL. The digestion reaction was carried out at 37°C for 4 h. The digested target fragment was recovered by tapping the rubber, and the digested ω3 FAD gene fragment was ligated with the pYES2 fragment with T ₄ DNA ligase to obtain the recombinant vector pY-ω3 FAD. The ligation reaction system was: 2.5 μL of buffer, about 0.3 pmol of ω3 FAD gene DNA fragment, about 0.03 pmol of DNA digested fragment of vector pYES2, 1 μL of _T4 DNA ligase, and RNase-free water to 25 μL. Ligation overnight at 16°C. After ligation, Escherichia coli DH5α competent cells were transformed, and cloning, colony PCR verification and sequencing were carried out according to the above method. The pY-ω3 FAD plasmid was extracted from Escherichia coli DH5α and stored at -20°C for future use.

(8) Preparation of yeast competent cells. The yeast was inoculated in YPD medium, revived at 28°C overnight, then scaled up at 1:100, and cultured with shaking at 250 rpm until the cell density was about 1×10 ⁸ cells/mL (about 4-5 h). Cool on ice for 15 min to stop the cell growth, collect yeast cells by centrifugation at 5 000 rpm for 5 min at 4°C, wash the cells twice with pre-cooled sterile water, and collect by centrifugation under the same conditions. Wash the cells once with 20 mL of pre-cooled 1 M sorbitol, then dissolve in 0.5 mL of pre-cooled 1 M sorbitol, and adjust the cell concentration to 1×10 ¹⁰ cells/mL. Store cells on ice (or 4°C) for electroporation.

(9) The electroporator (Bio-Rad) was electroporated, and the recombinant vector pY-ω3 FAD was transformed into yeast competent cells. Take about 5-10 μL (5-200 ng) of the pY-ω3 FAD recombinant plasmid DNA to be transformed that was pre-cooled on ice and mix it with the competent cells, and pre-cool on ice with a 0.2 cm electric shock cup, and then put Transfer the DNA-cell mixture to a pre-cooled electric shock cup and mix gently. After 5 min of ice bath, select program Sc2 to shock once, remove the electric shock cup, immediately add 1 mL of pre-cooled 1M sorbitol, and gently transfer to a new In the YPD medium of YPD medium, shake slightly at 28°C for 5h, spread the bacterial solution on the uracil-deficient synthetic medium (SC-U) containing 1 M sorbitol, and culture it upside down at 28°C for 48-72h, pick Colonies were cultured in liquid SC-U medium. After colony PCR verification, the strains were preserved in SC-U medium containing 2% glucose or 2% raffinose.

(10) Inoculate the preserved bacterial liquid into the YPD medium, shake and cultivate at 220 rpm at 28°C. Then the bacterial liquid was collected by centrifugation, resuspended in YPD medium with 2% galactose, and the substrates LA (C18:2ω6) and ArA (C20:4ω6) were added respectively to a final concentration of 0.005% (170 μM-180 μM). And add surfactant 1% Tergitol type NP-40, shake culture at 10°C at 150 rpm for 72 h. The yeast cells were collected by centrifugation, washed three times with deionized water, and frozen in liquid nitrogen.

(11) Freeze-dry the yeast frozen in liquid nitrogen, and then perform fatty acid methylation. Weigh about 25 mg of yeast powder into an esterification bottle, and add 2 ml of methanol solution containing 2% sulfuric acid. After being filled with nitrogen, the esterification reaction was carried out in a water bath at 82°C for 1 h, so as to fully dissociate and methylate the combined fatty acid molecules. After the esterification reaction, add 1 mL of deionized water and 1 mL of n-hexane to the above-mentioned esterification bottle, mix well, and then centrifuge at 5500 rpm for 10 min. The supernatant was collected into a sample bottle, concentrated with nitrogen, and stored at 4°C for later use.

(12) Analyzing the composition of fatty acids with an Angilent 6890 plus gas chromatograph, the chromatographic column is a HP-5 capillary column (30 m×0.25 mm); the heating program is 50°C for 1 min, and the temperature is raised to 200°C at 25°C/min , and then heated up to 230°C at 3°C/min, and finally kept for 18 min; the split ratio was 50:1; the flow rates of nitrogen, hydrogen, and air were 30 mL/min, 450 mL/min, and 40 mL/min, respectively. The injection volume was 1 μL.

(13) GC-MS detection of the generated products. The chromatographic column of GC is HP-INNOWax Polyethylene Glyco (30 m×320 μm×0.25 μm), connected to MS quadrupole, 150 C (maximum 200 C). The heating program is 50°C for 1 min, 10°C/min to 150°C and then 1 min, then 4°C/min to 230°C and then 5 min, and the running time is 37 min. Helium is the carrier gas, the initial value is 50°C, the pressure is 7.6522 psi, the flow rate is 2.6891 mL/min, the average velocity is 59.763 cm/s, the residence time is 0.83664 min, the flow rate is 2.6891 mL/min, and the running time is 37 min. The EMV mode was selected as the MS acquisition parameter, and the ion energy tracked and detected was 69.922 eV.

3. Results

(1) The ORF sequence of the ω3 FAD gene was amplified by the RACE method. The 5′- and 3′-terminal RACE amplification and the electrophoretic pattern of the specific target band are shown in Figure 1. In the figure, M stands for DL 2000 molecular weight standard; A picture shows the 5'-end product, the length is 650 bp; C picture shows the 3'-end product, the length is 1759 bp; B picture shows the target band, the length is 671 bp. The ORF sequence of the ω3 FAD gene obtained by sequencing is shown in SEQ ID NO.9. The full-length DNA sequence obtained by cloning and sequencing is shown in SEQ ID NO.10. The full-length structure of the ω3 FAD gene of Chlorella notch margin is shown in Figure 2.

和Xba I的基因操作，成功地构建了ω3 FAD基因的表达载体pY-ω3 FAD。将ω3 FAD基因组装在pYES2载体的GAL1启动子下游，应用半乳糖诱导基因过表达。 (2) Use the pYES2 vector to pass the restriction endonuclease Eco R

Gene manipulation with Xba I successfully constructed the expression vector pY-ω3 FAD of ω3 FAD gene. The ω3 FAD gene was assembled downstream of the GAL1 promoter of the pYES2 vector, and galactose was used to induce gene overexpression.

(3) The recombinant expression vector pY-ω3 FAD was successfully introduced into yeast INVSc1 by electroporation. Since the yeast INVSc1 is a His ^– , Leu ^– , Trp ^– , Ura ^– deficient strain, and the pYES2 vector has the URA3 gene encoding uracil, the yeast transformed with the pY-ω3 FAD plasmid can grow in the uracil-deficient synthetic medium (SC-U), and then screened to obtain transgenic yeast cells.

(4) Exogenous substrates LA and ArA were added to the culture medium, and after induction by galactose, GC-MS was used to detect the methylated fatty acids of yeast. The detection results of the yeast fatty acid components in the group without substrate addition are shown in Figure 3, and the arrows in the figure indicate the fatty acid components synthesized by the yeast itself; the detection results of the yeast fatty acid components in the group with the addition of substrate LA (C18:2 ^{Δ9, 12} ) are shown in Figure 4 As shown, the thick arrow in the figure indicates the added exogenous fatty acid substrate, and the thin arrow indicates its product. In Figure 3 and Figure 4, a represents the wild-type control group, b represents the empty vector control group, and c represents the transgenic yeast pY-ω3 FAD group containing ω3 FAD. The GC-MS spectrum is shown in Figure 5.

The content of fatty acid components in cells (% of total fatty acids) after the transgenic yeast was cultured with different fatty acid substrates was compared, and the results are shown in Table 1:

Table 1

It can be seen from Table 1 that the expression product of ω3 FAD gene can desaturate exogenous LA (C18:2ω6) into ALA (C18:3ω3) in yeast, and the conversion efficiency is 27.77%. No desaturation. These results indicated that the FAD encoded by the ω3 FAD gene cloned from Chlorella nicki has selectivity for substrates and can only desaturate LA (C18:2ω6) to ALA (C18:3ω3), thus the gene The encoded desaturase belongs to the Δ15 FAD of the ω3 FAD.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the method of the present invention, some improvements and supplements can also be made, and these improvements and supplements should also be considered Be the protection scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Ocean University

  ENN Technology Development Co., Ltd.

<120> A DNA Sequence Encoding Δ15 Fatty Acid Desaturase of Chlorella nickifolia and Its Application

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<160> 10

<170> PatentIn version 3.3

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gtgcctaaca aggacgtggg ttgtcccgac gtgcaaaccc ggcaacaatg caggccccca 120

ctatgtctac gacaggtgca ctgtatctgg gccgccctgt ggtgctgcgc gctggacact 180

cgcgccccag agccaatgcg cttttgcaga gcggtcggag gcaagcgttg cacgtgtgaa 240

catcgccgca gccccctaga ccagcctatg tcgacgaggt gagagctgaa ggcgctgaag 300

gacagcacgc tgttaatgcg tcgtaccact gcaaggagca cttggaggtg gtgtcgacgt 360

gacgttcagc aagcctcaac ctggctctgc gctgtcccaa gctgtttgct gggctcaagc 420

gtggcggcgg gcggatcata tggggctatg ttgactgctc ttggttggca acccctaatt 480

tgcactggca ctgatctacg acgcacctgc aggctgacta cagttgggct gcacaccgat 540

ctaacatcgg tgcataagtc agtgctagtg cttgcagaca tcgcttgctg aaacatgctt 600

tttgttggct gctgtcagac tggttatgga cgtgccgctc caccgccctt cacgctagct 660

gatatcaagg cagcagtgcc tgctcactgc tggaagaaga gcacgtggcg ctcgatggcc 720

ttcctggccc gcgatgtggg cgtggtggcg gcgctggctg tgggagccta cactctgaac 780

gcctggtgag cactttcata gcacaccctt ccagagtgct tttgacttga gctggggtca 840

aaggccacca catggaggtc ctcctggtta tgaaccacaa acagccctcc agttgcatgc 900

tgccaaggcg tcacgcaggc atgccgcttt tgcttgatgc agggtgcaca gctgcagctc 960

tcggtgctgt tctgactgta tgctgggtgg cacaggtggg cgtggccgct gtactgggtg 1020

gcacaggggga cgatgttctg ggccctcttt gtggtcgggc atgactggta aggccagata 1080

tatgcagctt tggaggcgat tgtcacatgc ctttgataag cgggcgtctg gaggcttggc 1140

ggtagctgag ccgtactgac acgccctgtt gttgtatgca gcggccatca gagcttctcc 1200

agcaacaaga cgctcaacaa cctggtgggc aacatcacac actcgtccat cctggtgccc 1260

taccacggct ggaggatcag ccaccgcaca catcatgcca accatggaca tgtcgagaac 1320

gatgagtcct ggcatcctgt cagcaagcgg atatacaacc agatggtgcg cagcgcttcc 1380

gcagtgaaac tagcagctgg gttccttggc attacctaag catgcctgca caatgtttcg 1440

atgacactgc tgctcgctgg agcggtagct agtgctgcgg atgagtgaag aagctgcggt 1500

gctctgcagg aatccatggc caagattggt cggctggcgt tcccgctgcc gctgtttgcc 1560

tacccctttt acctgtggca gcggtcgcct ggcaagacag gctcacacta tgaccccaag 1620

tgcgaccttt ttgtgccgca ggaagccccc atggtgaggc caggctcaat cttgtgattg 1680

tgcagctgcc aggatgtgga ttgccttggt gggctgttgt tgcccaagtg gctttgttga 1740

ttgcttagta ccttgtccat gccccatgcg tagctggcca ggtgtccttc tgttggcaat 1800

gccccagctc tttccacaga gtgatgccct gatgtctgtc atgctgcatg cagatccgca 1860

cgtccaacgc attcatgctg ggcatgctcg ccatcctggg cgcatgcaca tacgcgctgg 1920

gcccgctggc catgttcaac ctgtacttca tcccatacgt catcaacgtg gtctggctgg 1980

acgccgtcac ctacctgcat caccatgggc cgcatgacga gaacgagaag atcccctggt 2040

accgtggcga ggtgaggcga tggtgctacc ctttgaaata tgttggactt gctactggga 2100

tcttaacaac attcttgtat ggacggctgc tgtctgcaca acatcttggc gcagtgccat 2160

ttgctgcagc atcatgtcaa gctgacatgt tgggacctct gcgaattggg ccctaagccg 2220

tagtcgtctc gtggcaggag tggagctacc tgcgcggcgg cctgtccacc atcgaccgcg 2280

attttggcat cttcaaccac atccaccacg attggcac gcacgtgctg caccacctgt 2340

tccccccagat cccgcactac cacctggtgg aggcgaccga ggccgtcaag ccggtgtttg 2400

gcaactacta ccgcgagccc gagccctccc cgggccccat cccgacccat ctgttcaagc 2460

ccctcatgcg cagctttgcg caggaccact atgtcgagga tgagggcgac attgtgtact 2520

ataagactga ccccaagctg aaccatctgg ccagtggctc ggatgtgtag acggccccag 2580

cgcatgagcc caggtgcaag gcccgcaatg gctgggtcag cacataacg ggcagggcgc 2640

ccattcctgt ttgtcactgg gtcttgcgcg gataacaaaa catggcttgg aagcttgggg 2700

tcttcacggg gcgtgctgct ttagggttct atggcgcagg tcgccttggc caatagttgg 2760

agcagggcag gagctgtgtc ttgcggcttt accaaagagc agtgcagcag catgtgctgt 2820

cagcataggg taagggcggg cgtgctacca ataatgtgtt gcgctgccag tcgctgacat 2880

gtatcgggct gctgcacgag agtgcacagg tgtttgctgt ctctatgcca ggcagctttg 2940

tggttgcata gcgtagtccc aaacagtgca tgcaaccaat agctgcccag cgttgacttg 3000

tctggaagtg ctttcgggtt ctgattctga tcctgtcact gtcactgttg cagtcttctt 3060

agtctgctac ccagagacac agtgcagttt caagtgttca cccttagta ttctttctag 3120

cggcatctta gtggcggcct tggctaagcg gttgtgtcat tcaggtgttg tgtcgtggca 3180

caccaggcat gtctggcggg gttggctccc aacaaatgat gttctgtcaa ggtgttgatt 3240

ctttacgcta ttctttcggc ctctttaggt catgtctgat tggactgtga cagtgttagt 3300

ccagagttat gtacatcatt cgctctgccg gttcacatgc taagctggtg atctaggtct 3360

agcatcaaag cgttactggt gcgacttggt agacaagaac agcatctcgc tttgtgtcat 3420

ttgtaacatc agaacgcctc cgt 3443

Claims (9)

1. A DNA sequence encoding the Δ15 fatty acid desaturase of green algae, characterized in that the DNA sequence comprises: (a) the nucleotide sequence set forth in SEQ ID NO.10, or (b) A nucleotide sequence complementary to the nucleotide sequence described in (a). 2. A recombinant expression vector, characterized in that said vector is a recombinant expression vector constructed from the nucleotide sequence according to claim 1 and a plasmid or virus. 3. The recombinant expression vector according to claim 2, wherein said plasmid is pYES2 plasmid. 4. A genetically engineered host cell, characterized in that said host cell is selected from the following host cells: (a) host cells transformed or transduced with the nucleotide sequence of claim 1 and their progeny cells; (b) host cells transformed or transduced with the recombinant expression vector of claim 2 or 3 and their progeny cells. 5. The host cell according to claim 4, characterized in that, the host cell is a bacterial cell, a fungal cell, a plant cell or an animal cell, or a progeny of these host cells. 6. The host cell according to claim 4, characterized in that, the host cell is a yeast cell. 7. The application of the DNA sequence according to claim 1, the recombinant expression vector according to claim 2 or 3, or the host cell according to claim 4 or 5 in the production of long-chain polyunsaturated fatty acids. 8. The application according to claim 1, characterized in that, the long-chain polyunsaturated fatty acid is ω3 long-chain polyunsaturated fatty acid. 9. The application according to claim 1, characterized in that, the long-chain polyunsaturated fatty acid is α-linolenic acid.

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