CN101289659B

CN101289659B - Delta6 fatty acid desaturated enzyme of marine microalgae and applications thereof

Info

Publication number: CN101289659B
Application number: CN2008101152453A
Authority: CN
Inventors: 俞建中; 潘克厚; 马晓磊; 杨官品; 于文功; 朱葆华
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2008-06-19
Filing date: 2008-06-19
Publication date: 2010-12-22
Anticipated expiration: 2028-06-19
Also published as: CN101289659A

Abstract

The invention provides marine microalgae delta6-fatty acid desaturase, and coding genes and application thereof. The delta6-fatty acid desaturase is provided with amino acid sequences shown in a sequence table SEQ ID NO.2 or amino acid sequences which are formed by replacement, shortage or addition of one amino acid residue or a plurality of amino acid residues and have the same functions. The delta6-fatty acid desaturase can effectively catalyze Linoleic Acid (LA, 18:2delta9, 12) to generate gamma- Linolenic Acid (GLA, 18:3delta6, 9, 12) and catalyze alpha-Linolenic Acid (ALA, 18:3delta9, 12, 15) to generate Stearidnoic Acid (SDA, 18:4delta6, 9, 12, 15).

Description

A kind of marine microalgae delta6 fatty acid desaturase and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to Δ 6 fatty acid desaturases, its encoding gene and the application of a kind of marine microalgae-little plan ball algae.

Background technology

Lipid acid is the monocarboxylic acid with hydrocarbon chain, plays an important role in many biological procedureses.Lipid acid is less to be existed with the free form, but is advanced in the various main lipid composition, as phosphatide and triacylglycerol by fatization.Lipid acid mainly is divided into two classes: saturated fatty acid and unsaturated fatty acids, the latter is divided into monounsaturated fatty acids and polyunsaturated fatty acid again, contains one or more cis-double bondss (C=C) in its hydrocarbon chain respectively.

The generation of unsaturated link(age) needs fatty acid desaturase (fatty acid desaturase) catalysis.Fatty acid desaturase is present in nearly all plant, animal and the microorganism, the C-C singly-bound in the fatty acid chain can be converted into C=C pair and be good for.According to its position difference of introducing two keys, desaturase can be divided into Δ 5, Δ 6, Δ 9, Δ 12 etc., they introduce two keys at the 5th, 6,9,12 the carbon atom place that fatty acid chain is begun by carboxyl carbon respectively.The enzyme that adds two keys between the carboxyl carbon of fatty acid chain and the 9th carbon atom front end desaturase (front-enddesaturase) that is otherwise known as mainly comprises Δ 4, Δ 5, Δ 6, Δ 8 desaturases.People and other Mammals lack Δ 12 (ω 6) and Δ 15 (ω 3) desaturase, can not generate linolic acid (linoleic acid, LA; 18:2 Δ 9,12) and alpha-linolenic acid (α-linolenic acid, ALA; 18:3 Δ 9,12,15), must from food, replenish.Higher plant generally can only be synthesized to linoleic acid plus linolenic acid owing to lack the front end desaturase, can not continue the synthetic more polyunsaturated fatty acid of long-chain.And certain micro-organisms, particularly fungi, marine microalgae etc. then have synthesis of long-chain polyunsaturated fatty acids (long chainpolyunsaturated fatty acids, LCPUFAs) required all desaturases, can de novo synthesis LCPUFAs and content abundant (Sayanova and Napier, 2004).

LCPUFAs is meant the lipid acid (Abbadi et al., 2004) that contains 20 or 22 carbon atoms and 4-6 methene cis-double bonds at interval, comprises AA (arachidonic acid, 20:4 Δ 5,8,11,14), EPA (eicosapentaenoic acid, 20:5 Δ 5,8,11,14,17) and DHA (docosapentaenoic acid, 22:6 Δ 4,7,10,13,16,19) (be commonly called as " DHA (docosahexaenoic acid) ") etc.LCPUFAs can be divided into n6 series and n3 series according to last two bond length in the fatty acid chain from the distance of methyl end carbon atom.AA belongs to n6 LCPUFAs, and EPA and DHA belong to n3 LCPUFAs.Studies show that LCPUFAs has very important influence to human health.AA and EPA are the components of mammalian cell membrane, also are the precursors (Funk, 2001) that generates hormones such as prostaglandin(PG), leukotriene and thromboxane; EPA in various physiological responses such as blood coagulation, immunity and anti-inflammatory, play an important role (Simopoulos, 2002); DHA is to most important (the Uauy et al. of the formation of fetal nerve system, 2001), also affect activity (the Giusto et al. of retina Visual purple, 2000), and with prevention and treatment relevant (Horrocks and Yeo, 1999 of some disease such as sacroiliitis, arteriosclerosis, dysthymia disorders; Marszalek and Lodish, 2005).

AA, EPA and DHA are generated through a series of desaturations and extension by LA and ALA in vivo.At first LA and ALA generate GLA (18:3 Δ 6,9,12) and SDA (18:4 Δ 6,9,12,15) respectively under Δ 6 desaturase effects; Generate DGLA (20:3 Δ 8,11,14) and ETA (20:4 Δ 8,11,14,17) through Δ 6 extensions then; Generate AA (20:4 Δ 5,8,11,14) and EPA (20:5 Δ 5,8,11,14,17) by Δ 5 desaturase effects again; EPA can further generate DHA through two approach again.Though human body can be converted into AA, EPA and DHA with external source LA and ALA, combined coefficient is extremely low, can not satisfy the demand far away, takes in competent LCPUFAs (particularly EPA and DHA) and just seem particularly important in diet.

The source of current EPA and DHA mainly is a fish oil, yet along with the minimizing day by day with ocean harvestable fish class resource of increasing rapidly of the market requirement, this approach can not be satisfied the demand far away.In addition, reason such as environmental pollution causes the heavy metal content in the fish oil more and more higher.Therefore, seek more lasting, stable EPA and DHA source and become the task of top priority (Tononet al., 2002; Domergue et al., 2005a).Though now developed business-like algal oil and fungal oil, output that it is lower and higher extraction cost have limited the large-scale application of this class resource.The extraction process of considering vegetables oil is very ripe, many investigators have turned to sight the metabolic engineering of plant, explore and how to utilize existing front end desaturase and elongase gene particularly to make up the synthetic path of LCPUFAs in the oil crops, make it to become stable, cheap LCPUFAs source higher plant.Present this field Journal of Sex Research progress that made a breakthrough, the render transgenic plant produces EPA, DHA become a reality (Truksa etal., 2006) as " green cell factory ".

Δ 6 desaturases energy catalysis Linoleic Acid (18:2 Δ 9,12) generate γ-Linolenic Acid (18:3 Δ 6,9,12), and catalysis α-Linolenic Acid (18:3 Δ 9,12,15) generate Stearidnoic Acid (18:4 Δ 6,9,12,15), be the key enzyme of EPA and DHA generation thereafter.From animal, plant, fungi and marine microalgae, clone Δ 6 delta 8 desaturase genes now and carried out Function Identification.Leading a cow on the winged petiole mountain as Cohoon etc. detects Δ 6 acyl-ACP desaturases in (Thunberia alata) seed endosperm, means that the 1st two keys of lipid acid also may be in 6 generations of Δ (Cohoon et al., 1994).Clone from marine microalgae Ostreococcus tauri such as Domergue obtains an acyl group-CoA Δ 6 desaturases, and LA that this endonuclease capable catalysis is connected with CoA and ALA generate GLA and SDA (Domergue et al., 2005).But the Δ 6 desaturase quantity of commercial applications that can effectively be used in transgenic plant at present are few, and range of choice is narrow and small, and therefore continuing Δ 6 desaturases that clone and screening have higher Substratspezifitaet and catalytic activity is one of current research focuses.

Known little plan ball algae is rich in EPA, and high-content can reach more than 30% of total fatty acids, and Δ 6 desaturases may have high enzyme as EPA synthetic key enzyme and live.But do not see the relevant report of relevant little plan ball algae Δ 6 desaturases at present.

Summary of the invention

The object of the present invention is to provide a kind of little plan ball algae Δ 6 delta 8 desaturase genes, its proteins encoded and application.

Little plan ball algae Δ 6 delta 8 desaturase genes of the present invention have the nucleotide sequence shown in the SEQ ID NO.1, and its proteins encoded has the aminoacid sequence shown in the SEQ ID NO.2; The present invention also comprises the aminoacid sequence shown in the SEQ ID NO.2 through replacing, lack or add one or several amino acids formed derived protein with same function, and these proteinic genes of encoding.

The long 1425bp of Δ 6 delta 8 desaturase genes of the present invention, 474 amino acid of encoding.Carrying out the homology search in GenBank, do not find the sequence report identical with this gene, is a new gene.

Gene of the present invention can obtain in the following way: the cDNA that obtains with the total RNA reverse transcription of little plan ball algae is a template, utilize following primer to increase, the condition of Forward Primer:5 '-ATGGGACGCGGTGGCGAGCGG-3 ' Reverse Primer:5 '-TTACATGGCGGGGAAATCGGCC-3 ' amplification is: 94 ℃ of pre-sex change 3min, with 94 ℃ of 30s, 56 ℃ of 30s, 30 circulations of 72 ℃ of 1.5min reactions, 72 ℃ are extended 15min, 4 ℃ of preservations.

The present invention also comprises the expression vector that contains said gene, the host cell that contains described expression vector.

Δ 6 desaturases of the present invention have the peculiar N terminal cell of front end desaturase pigment b ₅Structural domain and 3 Histidines bunch (176-180,215-219,413-417).

Δ 6 desaturases of the present invention are catalysis catalysis Linoleic Acid (18:2 Δ 9 effectively effectively, 12) generate γ-Linolenic Acid (18:3 Δ 6,9,12), and catalysis α-LinolenicAcid (18:3 Δ 9,12,15) generate Stearidnoic Acid (18:4 Δ 6,9,12,15).

Dna sequence dna of the present invention can be applied to comprise arbitrary expression system of animal, plant, microorganism, and the render transgenic cell generates LCPUFAs.For example, in the plant of oil crops flax (Linum sp.), rape (Brassica sp.), soybean (Glycine and Sola sp.), Sunflower Receptacle (Helianthus sp.), cotton (Gossypium sp.), corn (Zea mays), olive (Olea sp.), safflower (Carthamus sp.), cocoa (Theobroma cacca) and peanut (Arachis sp.) and seed, express this gene and Δ 5 desaturases, Δ 6 extends genes such as enzymes, can make above-mentioned plant generate EPA, DHA, have high business development and be worth.And EPA, DHA can be used as healthcare products or medicine is used for prevention and treatment of diseases, as diabetes, cancer, inflammatory reaction and cardiovascular disorder etc.

Description of drawings

Fig. 1 is Δ 6 fatty acid desaturase catalysis (18:2 Δs 9 of the present invention, 12) generate (18:3 Δ 6,9,12) chromatography of gases analytical results, wherein 1-A does not add the spectrum peak figure of substrate for changing sky pYES2 plasmid over to, and 1-B is the spectrum peak figure of the empty pYES2 plasmid of commentaries on classics of adding LA substrate, 1-C is the spectrum peak figure of the commentaries on classics pYND6 plasmid of adding LA substrate, and 1-D is standard control;

Fig. 2 is little plan ball algae Δ 6 fatty acid desaturase catalysis (18:3 Δs 9,12,15) generate (18:4 Δ 6,9,12,15) chromatography of gases analytical results, wherein 2-A does not add the spectrum peak figure of substrate for changing sky pYES2 plasmid over to, and 2-B is the spectrum peak figure of the empty pYES2 plasmid of commentaries on classics of adding ALA substrate, 2-C is the spectrum peak figure of the commentaries on classics pYND6 plasmid of adding ALA substrate, and 2-D is standard control.

Embodiment

Following embodiment be used for to of the present inventionly advance-go on foot explanation, but be not used for limiting the scope of the invention.

The acquisition of embodiment 1 Δ 6 saturated enzyme genes

Concrete steps comprise:

1, the cultivation of little plan ball algae

1) little plan ball algae Nannochloropsis.oculata (Droop) Hibberd for purebred cultivation, is numbered CS-179 available from the little algae germplasm of Australian CSIRO storehouse (CSIRO Collection of Living Microalgae)

2) cultivating with seawater is coastal waters, Qingdao nature seawater, and seawater filters through absorbent cotton, and 100 ℃ are boiled 3min.250ml Erlenmeyer flask and transfer pipet etc. all with preceding autoclaving (121 ℃, 15min).

3) preparation f/2 mother liquor and sterilization.

4) ratio in 1: 1000 (v/v) adds the f/2 mother liquor in the sterilization seawater, and the ratio access algae kind in 1: 10 (v/v) places illumination box not inflate cultivation, shakes bottle every day for several times.Concrete culture condition sees Table 1.

The culture condition of the little plan ball of table 1 algae

The preparation of f/2 mother liquor

1. fundamental element:

NaNO3 78.4g+1L DDW (distilled water);

NaSiO3·9H2O 10g+1L?DDW

NaH2PO4 4.4g+1L?DDW

Mentioned reagent autoclaving (121 ℃, 15 minutes) adds 1ml in every 1000ml nutrient solution.

2. micro-:

ZnSO4·4H2O 2.3g

MnCl2·4H2O 17.8g

CuSO4·5H2O 1g

Na2MoO4·2H2O 0.73g

CoCl2·6H2O 1.2g

Na2EDTA 5g

Mentioned reagent is dissolved among the 100mlDDW, gets the 1ml mixed solution and adds among the 1L DDW, and other adds 4.3 gram Na2EDTA, and the micro-mother liquor for preparing stores behind remaining mixed solution autoclaving.Add 1ml trace element mother liquor during use in every 1000ml nutrient solution.

3.Fe element

FeC6H5O7·5H2O 39g+100ml?DDW

Get 10ml and add among the 1L DDW, store with the rest solution autoclaving as mother liquor.Add the 1ml mother liquor during use in every 1000ml nutrient solution.Fe also can fit over micro-mother liquor, also is to add 10ml.

4. VITAMIN

VB12 0.5mg

VB1 100mg

VH 0.5mg

The bottled 1L DDW of brown reagent autoclaving adds said vitamin, 4 ℃ of storages after reducing to room temperature.Add 1ml VITAMIN mother liquor during use in every 1000ml nutrient solution.

4) observe the growing state of algae every day, algae liquid yellow-green colour during little plan ball algae normal growth, but the frond long period be suspended in the substratum, microscopy is carried out in the cultivation of regularly going down to posterity when going down to posterity, observe to have or not frond adhesion or harmful organisms to pollute.

2, mRNA extracts

MRNA extraction and application TR1zol reagent (Invitrogen, Cat.No.15596-026) cracking, be used in combination RANprep Plant Kit (TIANGEN, Cat.No.DP402-02).Carry out the RNA purifying, detailed process is seen the reagent working instructions.

3, library construction

1) cDNA preparation:

Utilize SMART ^TMPCR cDNA Synthesis Kit (Clontech, Cat.No.634902), preparation cDNA, detailed process is seen the reagent working instructions.

2) the cDNA segmentation is reclaimed, is connected:

1. get the homogenization cDNA product electrophoresis of 200 μ l secondaries amplifications, reclaim 0.5-1.0kb respectively, 1.0-1.5kb, 1.5-2.0kb and＞the purpose fragment of 2.0kb;

2. carrier and competence are used Promega company

Vector Systems and JM109 (Cat.#3610);

3. carrier is connected (T4DNA ligase) with the insertion fragment with about 1: 3 ratio.

3) heat shock transforms

1. get 4 μ L and connect product, add in the 100 μ L competent cells mixing, precooling 20min on ice;

2. 42 ℃ of heat shock 45s;

3. behind the cooled on ice 2min, add 0.9ml SOC liquid nutrient medium;

4. 70rpm, 37 ℃, recovery 60min;

5. get 5 μ L bacterium liquid and be coated with LB (Ampr) flat board, X-Gal, IPTG screening was cultivated 16 hours for 37 ℃.

4) cDNA library order-checking

Picking mono-clonal at random from the flat board, LB cultivates, and send associating Gene science (group) company limited (www.chinagenenet.com) to check order after PCR detects.

5) sequencing result is carried out the Blastx comparison in GenBank, find a sequence and hailian seaweed, Δ 6 delta 8 desaturase genes of little algae such as Phaeodactylum tricornutum have very high similarity, tentatively confirm as little plan ball algae Δ 6 delta 8 desaturase genes 3 ' sequence.

4,5 ' RACE obtains 5 ' sequence of Δ 6 delta 8 desaturase genes

Use 5 ' RACE test kit to be: 5 ' RACE System for Rapid Amplification ofcDNA Ends, Version 2.0 (Invitrogen, Cat.No.15590-101).In the experiment each reagent be if no special instructions test kit from carries product, detailed process sees the test kit working instructions for details.According to outer primer in following design 5 ' the RACE nest-type PRC of 3 ' sequences Design

ND (outward): 5 ,-GTTCATGGCCACGAAGTAGAGACC-3,

ND (interior): 5 ,-CAGTCGCGTACGAATGCCAGCAGA-3,

The fragment that obtains cuts obvious band behind the electrophoresis, T-A connects, and transforms, and the bacterial strain that is accredited as positive colony send company's order-checking.Order-checking identifies that the fragment that a length is about 900bp is Δ 6 fatty acid desaturase gene 5 ' sequences.

5, the acquisition of the full gene of Δ 6 fatty acid desaturases

1) according to 3 ' and 5 ' terminal sequence information, search the reading frame in the GenBank comparison, design following primer (two ends band Kpn I and Sac I restriction enzyme site):

ND-Kpn?I-F：

5′-CGGGTACCACCATGGCACGGGATGGCGAGCCGGTCG-3’

ND-Sac?I-R：

5′-GCGGAGCTCTTCGCTTCCCTCCGTCCGTTTACACAG-3’

The cDNA product that obtains during 2) with the structure library is a masterplate, and amplification obtains the full gene of Δ 6 fatty acid desaturases, and the PCR reaction system is as follows:

Sterilized，distilled?water 36.5μl

10×PCR?buffer 5.0μl

10mM?dNTP?mix 1.0μl

ND-Kpn?I-F(10μM) 1.0μl

ND-Sac?I-R(10μM) 1.0μl

cDNA 5.0μl

Cumulative volume _ 49.5 μ l

Add high-fidelity DNA polymerase (5U/ μ l) mixing immediately, following condition is carried out pcr amplification: 94 ℃, and 2m → (94 ℃, 30s → 55 ℃, 30s → 72 ℃, 1m) * and 35cycles → 72 ℃, 7min

Amplification has obtained the coding region of this gene, and connects into pGEM-T plasmid commentaries on classics JM109 preservation, and sequence verification obtains Δ 6 delta 8 desaturase genes, and its nucleotide sequence is shown in SEQ ID NO.1.

6, according to above-mentioned sequencing result, Δ 6 fatty acid desaturase mrna lengths from little plan ball algae are 1425bp, 474 amino-acid residues of encoding, its amino acid sequence coded is shown in sequence table SEQ ID NO.2, this sequence has the feature structure territory of front end desaturase, comprises N terminal cell pigment b5 structural domain and 3 Histidines bunch (176-180,215-219,413-417).

The structure of embodiment 2 expression vector pYND6 and the conversion of yeast saccharomyces cerevisiae

1, yeast saccharomyces cerevisiae expression vector pYES2 and yeast strain INVSc1 brief introduction

The experiment expression vector is selected pYES2 plasmid (Invitrogen for use, Cat.No.V825-20), long 5857bp, have the GAL1 promotor, this promotor is an induction type, have glucose (glucose) to transcribe when existing in substratum and be suppressed, removing glucose adding semi-lactosi then can inducible transcription.In addition, cell can also be grown in the substratum of raffinose (raffinose) as carbon source, and raffinose is neither induced and also do not suppressed to transcribe, and inducedvelocity is that the carbon source cultured cells is fast than beginning with glucose.

The genotype of yeast INVSc1 is MATa his3 Δ 1 leu2 trp1-289 ura3-52/MAT α his3 Δ 1 leu2 trp1-289 ura3-52, and phenotype is His ^-, Leu ^-, Trp ^-, Ura ^-Plasmid pYES2 has the URA3 gene, the minimal medium screening yeast transformant of available shortage uridylic.Other details are referring to the pYES2 specification sheets of Invitrogen.

2, the screening of the structure of expression vector pYND6 and positive colony

1) the positive bacterium colony of the JM109 that contains pGEM-Δ 6 delta 8 desaturase genes that obtains among the amplification cultivation embodiment 1.

2) extract the pGEM plasmid that has Δ 6 delta 8 desaturase genes according to ordinary method, utilize corresponding D NA restriction endonuclease (Kpn I/Sac I), from the pGEM plasmid that inserts goal gene, cut gene, utilize corresponding restriction endonuclease combination simultaneously, the pYES2 plasmid is cut into the line style double-stranded DNA.

The restriction endonuclease reaction reagent is pressed following proportioning:

nuclease-free?water 15μl

10×Buffer 2μl

DNA/ plasmid 1 μ l

Restriction endonuclease 2 μ l

Cumulative volume 20 μ l

37 ℃ of reactions are spent the night, and product is cut glue and reclaimed purifying

3) carrier connects

Delta 8 desaturase genes after restriction endonuclease handled with cut the pYES2 plasmid and mix with 2: 1 volume ratios, press following proportioning mixed reaction solution:

DNA﹠amp; Plasmid 10 μ l

10×Buffer 2μl

nuclease-free?water 6μl

T4?DNA?Ligase 2μl

Cumulative volume 20 μ l

More than 22 ℃ of reaction 1h, the 5 μ l reaction product heat shock methods of getting change JM109 over to, and coated plate is chosen mono-clonal and cultivated, bacterium liquid PCR check back sequence verification.

Thus, make up and finish expression vector pYND6, will contain the e. coli jm109 amplification cultivation of expression vector, and extract plasmid, yeast conversion is prepared in-20 ℃ of preservations.

3, the preparation of yeast saccharomyces cerevisiae competent cell and conversion.

The preparation of the yeast saccharomyces cerevisiae competent cell of this experiment and trans-utilization EasyCompTM conversion reagent box (Invitrogen, Catalog no.K5050-01), specific operation process is seen the test kit working instructions.

Yeast after the conversion evenly is applied to SC-U-flat board (placing 1h for dull and stereotyped prior 30 ℃), and 30 ℃ of thermostat containers are cultivated 3d; Picking yeast list bacterium colony is cultivated, and extracts zymic DNA, is PCR with specially primer ND-Kpn I-F and ND-Sac I-R and detects, and the contrast of commentaries on classics empty plasmid is set.It is the positive colony that changes the ND6 gene over to that PCR can access this yeast of the segmental expression of purpose, can be used for expressing experiment.

Abduction delivering and the fatty acid analysis of embodiment 3 yeast strains

1. abduction delivering

1) will contain the positive colony of recombinant plasmid and empty plasmid contrast bacterium and rule on SC-U (2%glucose) flat board once more, 30 ℃ are cultured to and grow bacterium colony;

2) choose mono-clonal and insert 3ml SC-U (2%glucose) liquid nutrient medium, 30 ℃ are shaken bacterium and spend the night;

3) it is centrifugal to get an amount of bacterium liquid, adds 3.6mlSC-U (2%glucose), to initial OD ₆₀₀About 0.2, add 400 μ l20%NP-40 (final concentration is 1%), add lipid acid substrate (LA, 18:2 Δ 9,12 and ALA, 18:3 Δ 9,12,15) simultaneously and, induce 72h for 20 ℃ to final concentration 25uM.

2. lipid acid extracts and analyzes

2.1 lipid acid extracts and esterification

1) the bacterium liquid 5000rpm after will inducing, the centrifugal collection of 3min;

2) liquid nitrogen grinding changes the dry powder after grinding in the EP pipe over to, adds the saturated methyl alcohol of 300 μ l KOH, 70-80 ℃ of saponification 15-20min after the vortex mixed;

3) add the HCl-methyl alcohol of excessive 1N concentration, vortex mixing, 70-80 ℃ of esterification 15min after the cooling;

4) add an amount of normal hexane, and drench a small amount of distilled water, the layering extraction;

5) behind the centrifugal 5min of 12000rpm, carefully get the upper strata organic layer, promptly can be used for gas phase analysis.

2.2 the gas-chromatography of lipid acid (GC) is analyzed

1) instrument: the 5890 SERIES II of Hewlett-Packard;

Carrier gas: N ₂Linear speed: 20cm/s; Splitting ratio: 10: 1

Flow: 2.2ml/min; Temperature of vaporization chamber: 260 ℃;

Temperature programming: 150 ℃ of (2min) → 15 ℃/min rises to 200 ℃ of (0min) → 2 ℃/min and rises to 250 ℃ (15min);

Detector: flame ionization ditector (FID);

2) be standard substance behind the various methyl esterification of fatty acid with the production of Cayman company;

3) the lipid acid sample with standard substance and above-mentioned esterification carries out the GC analysis, and applied sample amount is 1-2 μ l.

4) analysis software: SPSIII type chromatographic data treatment system.

3. experimental result

Gas chromatographic analysis shows, change lipid acid substrate Linoleic Acid (the 18:2 Δ 9 that external source can be added behind the yeast abduction delivering of pYND6 over to, 12) catalysis generates γ-Linolenic Acid (18:3 Δ 6,9,12), and catalysis α-Linolenic Acid (18:3 Δ 9,12,15) generate Stearidnoic Acid (18:4 Δ 6,9,12,15).The albumen that institute's transgenes encoding is described has the function of Δ 6 fatty acid desaturases really, confirms as Δ 6 fatty acid desaturases of little plan ball algae.

Gas chromatographic analysis such as Fig. 1, shown in 2, Fig. 1 is result behind the adding LA, wherein 1-A does not add the spectrum peak figure of substrate for changing sky pYES2 plasmid over to, and 1-B is the spectrum peak figure of the empty pYES2 plasmid of commentaries on classics of adding LA substrate, 1-C is the spectrum peak figure of the commentaries on classics pYND6 plasmid of adding LA substrate, and 1-D is standard control.The interference at peak though have powerful connections, but can see significantly that the ND6 gene can obtain activity expression in yeast, catalysis LA generates DLA, and blank does not have this function.

Fig. 2 is for adding ALA result.Wherein 2-A does not add the spectrum peak figure of substrate for changing sky pYES2 plasmid over to, and 2-B is the spectrum peak figure of the empty pYES2 plasmid of commentaries on classics of adding ALA substrate, and 2-C is the spectrum peak figure of the commentaries on classics pYND6 plasmid of adding ALA substrate, and 2-D is standard control.The interference at peak though have powerful connections, but can see significantly that the ND6 gene can obtain activity expression in yeast, catalysis ALA generates SDA (the spectrum peak of SDA is than the obvious height of background peaks), and blank does not have this function.

N6 transformation efficiency ratio:

18:2 Δ 9,12 and 18:3 Δ 6,9,12 shared per-cent in analysis chart are respectively 6.63 and 3.92, and its transformation ratio is: 6.63+3.92

18:3Δ6，9，12/(18:2Δ9，12+18:3Δ6，9，12)

＝3.92/(6.63+3.92)＝37％

N3 transformation efficiency ratio:

(18:3 Δ 9,12,15) and (18:4 Δ 6,9,12,15) shared per-cent is respectively 7.74 and 5.86 in analysis chart, and its transformation ratio is:

(18:4Δ6，9，12，15)/(18:3Δ9，12，15+18:4Δ6，9，12，15)

＝5.86/(7.74+5.86)＝43％

Reference:

Domergue?F,?Lerchl?J，Zahringer?U，Heinz?E(2002).Cloning?and?functional?characterization?ofPhaeodactylum?tricornutum?front-end?desaturases?involved?in?dicosapenaenoic?acidbiosynhesis.Eur?J?Biochem?269，4105-4113.

Abbadi?A，Domergue?F，Bauer?J，Napier?JA，Welti?R，Zahringer?U，Cirpus?P，Heinz?E(2004).Biosynthesis?of?very?long-chain?polyunsaturated?fatty?acids?in?transgenic?oilseeds：constraints?ontheir?accumulation.Plant?Cell?16，2734-2748.

Cahoon?EB，Cranmer?AM，Shanklin?J?and?Ohlrogge?JB.1994.Hexadecenoic?acid?is?synthesized?bythe?activity?of?a?soluble?Δ6-palmitoyl-acyl?carrier?protein?desaturase?in?Thunbergia?alataendosperm.J.Biol.Chem.269：27519-26

Domergue?F，Abbadi?A，Heinz?E(2005a).Relief?for?fish?stocks：oceanic?fatty?acids?in?transgenicoilseeds.Trends?Plant?Sci?10，112-116.

Funk?CD(2001).Prostaglandins?and?leukotrienes：advances?in?eicosanoid?biology.Science?294，1871-1875.

Giusto?NM，Pasquare?SJ，Salvador?PI，Roque?ME，Ilincheta?de?Boschero?MG?2000.Lipidmetabolism?in?vertebrate?retinal?rod?outer?segments.Prog.Lipid?Res?39，315-319.

Horrocks?LA，Yeo?YK(1999).Healthbenefits?of?docosahexaenoic?acid(DHA).Pharmacol?Res?40，211-215.

Marszalek?JR，Lodish?HF(2005).Docosahexaenoic?acid，fatty?acid-interacting?proteins，andneuronal?function：breastmilk?and?fish?are?good?for?you.Annu?Rev?Cell?Dev?Biol?21，633-657.

Qi?B，Fraser?T，Mugford?S，Dobson?G，Sayanova?O，Butler?J，Napier?JA，Lazarus?CM(2004).Production?of?very?long?chain?polyunsaturated?omega-3?and?omega-6?fatty?acids?in?plants.NatBiotechnol?22，739-745.

Sayanova?O，Napier?JA(2004).Eicosapentaenoic?acid：biosynthetic?routes?and?the?potential?forsynthesis?in?transgenic?plants.Phytochemistry?65，147-158.

Simopoulos?AP(2002).Omega-3?fatty?acids?in?inflammation?and?autoimmune?diseases.J?Am?CollNutr?21，495-505.

Tonon?T，Larson?TR，Graham?IA(2002).Long?chain?polyunsaturated?fatty?acid?production?andpartitioning?to?triacylglycerols?in?four?microalgae.Phytochemistry?61，15-24.

Truksa?M，Wu?G，Vrinten?V，Qiu?X(2006).Metabolic?engineering?of?plants?to?produce?verylong-chain?polyunsaturated?fatty?acids.Transgenic?Res?15，131-137.

Uauy?R，Hoffman?DR，Peirano?P，Birch?DG，Birch?EE(2001).Essential?fatty?acids?in?visual?andbrain?development.Lipids?36，885-895.

Wu?G，Truksa?M，Datla?N，Vrinten?P，Bauer?J，Zank?T，Cirpus?P，Heinz?E，Qiu?X(2005).Stepwise?engineering?to?produce?high?yields?of?very?long-chain?polyunsaturated?fatty?acids?inplants.Nat?Biotechnol?23，1013-1017.

Sequence table

＜110〉Chinese Marine University

＜120〉a kind of marine microalgae delta6 fatty acid desaturase and application thereof

<130>

<160>8

<170>PatentIn?version?3.3

<210>1

<211>1425

<212>DNA

<213>Nannochloropsis.oculata

<220>

<221>CDS

<222>(1)..(1425)

<400>1

atg?gca?cgg?gat?ggc?gag?ccg?gtc?gag?acg?acg?gag?tct?ttg?agc?ttc 48

Met?Ala?Arg?Asp?Gly?Glu?Pro?Val?Glu?Thr?Thr?Glu?Ser?Leu?Ser?Phe

1 5 10 15

acg?gcc?gac?aag?gcg?ggg?acc?atc?aag?cag?cgt?ggg?cgg?aag?atc?aca 96

Thr?Ala?Asp?Lys?Ala?Gly?Thr?Ile?Lys?Gln?Arg?Gly?Arg?Lys?Ile?Thr

20 25 30

tgg?gat?gag?gtg?cgt?cag?cac?aag?acg?cct?cag?gac?gct?tgg?ctc?gtg 144

Trp?Asp?Glu?Val?Arg?Gln?His?Lys?Thr?Pro?Gln?Asp?Ala?Trp?Leu?Val

35 40 45

tat?agg?aat?aag?gtc?tac?gac?gtg?tcg?agc?tgg?caa?gat?cac?ccc?ggg 192

Tyr?Arg?Asn?Lys?Val?Tyr?Asp?Val?Ser?Ser?Trp?Gln?Asp?His?Pro?Gly

50 55 60

ggg?aac?gtc?atc?ttc?act?cac?gcc?ggc?ggg?gac?tgc?acg?gat?att?ttc 240

Gly?Asn?Val?Ile?Phe?Thr?His?Ala?Gly?Gly?Asp?Cys?Thr?Asp?Ile?Phe

65 70 75 80

gcg?gcg?ttc?cac?cct?ctt?ggc?gcc?acc?tct?tat?ctt?gat?cca?ttt?tac 288

Ala?Ala?Phe?His?Pro?Leu?Gly?Ala?Thr?Ser?Tyr?Leu?Asp?Pro?Phe?Tyr

85 90 95

att?ggc?gag?ctg?gag?ccg?cgc?tcg?gac?aag?aag?ccc?gca?gcg?cag?gcg 336

Ile?Gly?Glu?Leu?Glu?Pro?Arg?Ser?Asp?Lys?Lys?Pro?Ala?Ala?Gln?Ala

100 105 110

aac?ttt?gag?cgc?gcc?tac?agg?gat?ctc?agg?ggg?aag?ctt?atc?gcg?ggt 384

Asn?Phe?Glu?Arg?Ala?Tyr?Arg?Asp?Leu?Arg?Gly?Lys?Leu?Ile?Ala?Gly

115 120 125

ggg?ttt?ttc?aag?gcg?aat?cct?ttg?tac?tat?gtc?tgg?aag?gta?gta?tcg 432

Gly?Phe?Phe?Lys?Ala?Asn?Pro?Leu?Tyr?Tyr?Val?Trp?Lys?Val?Val?Ser

130 135 140

tca?gtt?gcc?ctt?gct?gta?ggt?gcg?tgg?gtg?ctg?gtg?gct?tgg?tcg?cag 480

Ser?Val?Ala?Leu?Ala?Val?Gly?Ala?Trp?Val?Leu?Val?Ala?Trp?Ser?Gln

145 150 155 160

aac?ctg?ggc?gtg?cag?atg?ctg?tct?gcg?ttt?ttg?gtg?gct?ctg?ttc?tgg 528

Asn?Leu?Gly?Val?Gln?Met?Leu?Ser?Ala?Phe?Leu?Val?Ala?Leu?Phe?Trp

165 170 175

cag?caa?tgt?ggc?tgg?ttg?gcc?cat?gac?ttc?ctg?cac?cac?cag?gta?ttt 576

Gln?Gln?Cys?Gly?Trp?Leu?Ala?His?Asp?Phe?Leu?His?His?Gln?Val?Phe

180 185 190

aag?aac?cgt?gcg?ttg?ggt?gac?ctg?gcc?ggc?atc?gtt?atc?ggc?aat?gtc 624

Lys?Asn?Arg?Ala?Leu?Gly?Asp?Leu?Ala?Gly?Ile?Val?Ile?Gly?Asn?Val

195 200 205

ttc?cag?ggt?ttc?tcc?gtg?gca?tgg?tgg?aag?aac?aag?cat?aac?act?cac 672

Phe?Gln?Gly?Phe?Ser?Val?Ala?Trp?Trp?Lys?Asn?Lys?His?Asn?Thr?His

210 215 220

cac?gcg?gtg?ccc?aac?ctc?gtc?gag?tcc?tct?ccg?gac?gcg?caa?gac?gca 720

His?Ala?Val?Pro?Asn?Leu?Val?Glu?Ser?Ser?Pro?Asp?Ala?Gln?Asp?Ala

225 230 235 240

gac?cct?gac?att?gac?acc?atg?ccc?ata?ctg?gcc?tgg?tcg?ctc?aag?atg 768

Asp?Pro?Asp?Ile?Asp?Thr?Met?Pro?Ile?Leu?Ala?Trp?Ser?Leu?Lys?Met

245 250 255

gcc?gac?agg?gcg?cag?caa?tac?tca?tgg?gga?ccc?ttc?ttt?gtc?agg?cat 816

Ala?Asp?Arg?Ala?Gln?Gln?Tyr?Ser?Trp?Gly?Pro?Phe?Phe?Val?Arg?His

260 265 270

cag?tcg?ctg?cta?tac?ttc?ccc?atc?ctg?ctc?gtg?gcg?cgg?att?tca?tgg 864

Gln?Ser?Leu?Leu?Tyr?Phe?Pro?Ile?Leu?Leu?Val?Ala?Arg?Ile?Ser?Trp

275 280 285

ttg?atg?cag?tcg?ttc?ttg?ttt?gtc?ttt?gac?tcc?gtc?cct?gga?gcg?agt 912

Leu?Met?Gln?Ser?Phe?Leu?Phe?Val?Phe?Asp?Ser?Val?Pro?Gly?Ala?Ser

290 295 300

ctg?tgg?gca?acc?aag?ggc?gcg?acg?gct?gag?aga?cag?gcg?atc?aag?aat 960

Leu?Trp?Ala?Thr?Lys?Gly?Ala?Thr?Ala?Glu?Arg?Gln?Ala?Ile?Lys?Asn

305 310 315 320

gtc?ggg?ttg?gag?aag?gtg?ggg?ctg?gtt?gcg?cac?tac?ctg?tgg?tac?ggt 1008

Val?Gly?Leu?Glu?Lys?Val?Gly?Leu?Val?Ala?His?Tyr?Leu?Trp?Tyr?Gly

325 330 335

gcg?ctg?atg?ctg?tgc?cac?atg?tcc?ctg?gcc?cgc?gcc?ctg?ctg?tac?ttc 1056

Ala?Leu?Met?Leu?Cys?His?Met?Ser?Leu?Ala?Arg?Ala?Leu?Leu?Tyr?Phe

340 345 350

ctg?ccg?agc?cag?atg?atg?tgc?ggg?ttc?ttg?ctc?gcg?ctt?gtt?ttc?ggg 1104

Leu?Pro?Ser?Gln?Met?Met?Cys?Gly?Phe?Leu?Leu?Ala?Leu?Val?Phe?Gly

355 360 365

ctt?ggg?cac?aac?ggc?atg?gat?gtt?tac?gac?gcg?gac?gcc?cgg?ccc?gac 1152

Leu?Gly?His?Asn?Gly?Met?Asp?Val?Tyr?Asp?Ala?Asp?Ala?Arg?Pro?Asp

370 375 380

ttc?tgg?aag?ctg?cag?gtg?acg?acg?acg?agg?aac?gtg?acg?ggc?tcg?tgg 1200

Phe?Trp?Lys?Leu?Gln?Val?Thr?Thr?Thr?Arg?Asn?Val?Thr?Gly?Ser?Trp

385 390 395 400

ttg?gtg?cag?tgg?ttc?tgt?ggc?ggc?ctc?ggc?tac?cag?gtg?gac?cac?cac 1248

Leu?Val?Gln?Trp?Phe?Cys?Gly?Gly?Leu?Gly?Tyr?Gln?Val?Asp?His?His

405 410 415

ctg?ttc?ccc?atg?atc?ccg?cgg?cac?cgc?cta?ggg?aag?ctc?cac?ggg?ctc 1296

Leu?Phe?Pro?Met?Ile?Pro?Arg?His?Arg?Leu?Gly?Lys?Leu?His?Gly?Leu

420 425 430

gtg?gag?ggt?ttc?tgc?aag?gat?cac?ggg?gtg?aag?tac?cac?gag?acg?aat 1344

Val?Glu?Gly?Phe?Cys?Lys?Asp?His?Gly?Val?Lys?Tyr?His?Glu?Thr?Asn

435 440 445

atg?tgg?gag?ggg?acc?aaa?gag?gtg?ttg?gct?cac?ttg?agc?agt?gtg?acg 1392

Met?Trp?Glu?Gly?Thr?Lys?Glu?Val?Leu?Ala?His?Leu?Ser?Ser?Val?Thr

450 455 460

aaa?gag?ttc?gtg?gcc?gat?ttc?gcc?gct?gtg?taa 1425

Lys?Glu?Phe?Val?Ala?Asp?Phe?Ala?Ala?Val

465 470

<210>2

<211>474

<212>PRT

<213>Nannochloropsis.oculata

<400>2

Met?Ala?Arg?Asp?Gly?Glu?Pro?Val?Glu?Thr?Thr?Glu?Ser?Leu?Ser?Phe

1 5 10 15

Thr?Ala?Asp?Lys?Ala?Gly?Thr?Ile?Lys?Gln?Arg?Gly?Arg?Lys?Ile?Thr

20 25 30

Trp?Asp?Glu?Val?Arg?Gln?His?Lys?Thr?Pro?Gln?Asp?Ala?Trp?Leu?Val

35 40 45

Tyr?Arg?Asn?Lys?Val?Tyr?Asp?Val?Ser?Ser?Trp?Gln?Asp?His?Pro?Gly

50 55 60

Gly?Asn?Val?Ile?Phe?Thr?His?Ala?Gly?Gly?Asp?Cys?Thr?Asp?Ile?Phe

65 70 75 80

Ala?Ala?Phe?His?Pro?Leu?Gly?Ala?Thr?Ser?Tyr?Leu?Asp?Pro?Phe?Tyr

85 90 95

Ile?Gly?Glu?Leu?Glu?Pro?Arg?Ser?Asp?Lys?Lys?Pro?Ala?Ala?Gln?Ala

100 105 110

Asn?Phe?Glu?Arg?Ala?Tyr?Arg?Asp?Leu?Arg?Gly?Lys?Leu?Ile?Ala?Gly

115 120 125

Gly?Phe?Phe?Lys?Ala?Asn?Pro?Leu?Tyr?Tyr?Val?Trp?Lys?Val?Val?Ser

130 135 140

Ser?Val?Ala?Leu?Ala?Val?Gly?Ala?Trp?Val?Leu?Val?Ala?Trp?Ser?Gln

145 150 155 160

Asn?Leu?Gly?Val?Gln?Met?Leu?Ser?Ala?Phe?Leu?Val?Ala?Leu?Phe?Trp

165 170 175

Gln?Gln?Cys?Gly?Trp?Leu?Ala?His?Asp?Phe?Leu?His?His?Gln?Val?Phe

180 185 190

Lys?Asn?Arg?Ala?Leu?Gly?Asp?Leu?Ala?Gly?Ile?Val?Ile?Gly?Asn?Val

195 200 205

Phe?Gln?Gly?Phe?Ser?Val?Ala?Trp?Trp?Lys?Asn?Lys?His?Asn?Thr?His

210 215 220

His?Ala?Val?Pro?Asn?Leu?Val?Glu?Ser?Ser?Pro?Asp?Ala?Gln?Asp?Ala

225 230 235 240

Asp?Pro?Asp?Ile?Asp?Thr?Met?Pro?Ile?Leu?Ala?Trp?Ser?Leu?Lys?Met

245 250 255

Ala?Asp?Arg?Ala?Gln?Gln?Tyr?Ser?Trp?Gly?Pro?Phe?Phe?Val?Arg?His

260 265 270

Gln?Ser?Leu?Leu?Tyr?Phe?Pro?Ile?Leu?Leu?Val?Ala?Arg?Ile?Ser?Trp

275 280 285

Leu?Met?Gln?Ser?Phe?Leu?Phe?Val?Phe?Asp?Ser?Val?Pro?Gly?Ala?Ser

290 295 300

Leu?Trp?Ala?Thr?Lys?Gly?Ala?Thr?Ala?Glu?Arg?Gln?Ala?Ile?Lys?Asn

305 310 315 320

Val?Gly?Leu?Glu?Lys?Val?Gly?Leu?Val?Ala?His?Tyr?Leu?Trp?Tyr?Gly

325 330 335

Ala?Leu?Met?Leu?Cys?His?Met?Ser?Leu?Ala?Arg?Ala?Leu?Leu?Tyr?Phe

340 345 350

Leu?Pro?Ser?Gln?Met?Met?Cys?Gly?Phe?Leu?Leu?Ala?Leu?Val?Phe?Gly

355 360 365

Leu?Gly?His?Asn?Gly?Met?Asp?Val?Tyr?Asp?Ala?Asp?Ala?Arg?Pro?Asp

370 375 380

Phe?Trp?Lys?Leu?Gln?Val?Thr?Thr?Thr?Arg?Asn?Val?Thr?Gly?Ser?Trp

385 390 395 400

Leu?Val?Gln?Trp?Phe?Cys?Gly?Gly?Leu?Gly?Tyr?Gln?Val?Asp?His?His

405 410 415

Leu?Phe?Pro?Met?Ile?Pro?Arg?His?Arg?Leu?Gly?Lys?Leu?His?Gly?Leu

420 425 430

Val?Glu?Gly?Phe?Cys?Lys?Asp?His?Gly?Val?Lys?Tyr?His?Glu?Thr?Asn

435 440 445

Met?Trp?Glu?Gly?Thr?Lys?Glu?Val?Leu?Ala?His?Leu?Ser?Ser?Val?Thr

450 455 460

Lys?Glu?Phe?Val?Ala?Asp?Phe?Ala?Ala?Val

465 470

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<400>3

atgggacgcg?gtggcgagcg?g 21

<210>4

<211>22

<212>DNA

＜213〉artificial sequence

<400>4

ttacatggcg?gggaaatcgg?cc 22

<210>5

<211>24

<212>DNA

＜213〉artificial sequence

<400>5

gttcatggcc?acgaagtaga?gacc 24

<210>6

<211>24

<212>DNA

＜213〉artificial sequence

<400>6

cagtcgcgta?cgaatgccag?caga 24

<210>7

<211>36

<212>DNA

＜213〉artificial sequence

<400>7

cgggtaccac?catggcacgg?gatggcgagc?cggtcg 36

<210>8

<211>36

<212>DNA

＜213〉artificial sequence

<400>8

gcggagctct?tcgcttccct?ccgtccgttt?acacag 36

Claims

1. A marine microalgae Δ6 fatty acid desaturase, the amino acid sequence of which is as shown in SEQ ID NO.2.

2. The gene encoding the marine microalgae Δ6 fatty acid desaturase of claim 1.

3. the gene as claimed in claim 2, its nucleotide sequence is as shown in SEQ ID NO.1.

4. An expression vector containing the gene of claim 2 or 3.

5. A host cell containing the expression vector of claim 4.

6. the application of the enzyme described in claim 1 in preparation EPA.

7. the application of the enzyme described in claim 1 in preparing DHA.