CN101210234B - Ocean micro-alga delta5 aliphatic acid desaturase and application thereof - Google Patents

Abstract

The invention provides a marine microalgae Δ5 fatty acid desaturase, its coding gene and application. The Δ5 fatty acid desaturase of the present invention has the amino acid sequence shown in SEQ ID NO.2 in the sequence table or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or several amino acid residues. The Δ5 fatty acid desaturase of the present invention can effectively catalyze ETA (20:4Δ8, 11, 14, 17) to generate EPA (20:5Δ5, 8, 11, 14, 17), and has high substrate specificity and enzymatic activity high.

Description

A kind of marine microalgae Δ 5 fatty acid desaturases and application thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of Δ 5 fatty acid desaturases, its encoding gene and application of marine microalgae-Phaeodactylum tricornutum.

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-end desaturase) 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 chain polyunsaturated fatty acids, LCPUFAs) required all desaturases, can de novo synthesis LCPUFAs and content abundant (Sayanovaand 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 n6LCPUFAs, and EPA and DHA belong to n3LCPUFAs.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 andLodish, 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 (Tonon et 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 et al., 2006) as " green cell factory ".

Δ 5 desaturases belong to the front end desaturase, and catalysis ETA (20:4 Δ 8,11,14,17) generates EPA (20:5 Δ 5,8,11,14,17), are the key enzymes of EPA and DHA generation thereafter.From animal, fungi and marine microalgae, clone Δ 5 delta 8 desaturase genes now and carried out Function Identification.Wherein the gene of non-animal-origin is applied to plant metabolism engineering (Qi et al, 2004; Abbadi et al., 2004; Wu et al., 2005) Δ 5 desaturases that, comprise Mortierella alpina (Mortierella alpine), Phaeodactylum tricornutum (Phaeodactylum tricornutum) and a kind of fungi (Thraustochytrium sp.).But the Substratspezifitaet of these several enzymes is relatively poor, except target product, also can generate some by product, as Δ 5 desaturases of Phaeodactylum tricornutum to 18:1 Δ 11,20:1 Δ 11,20:2 Δ 11,14,20:3 Δ 8,11,14, the 20:3 Δ 11, several lipid acid such as 14,17 all work (Domergue et al., 2002) (GenBank Accession No.AY082392).Because these by products are known little to the influence of transgenic plant and human body, their appearance is unfavorable for the commercial applications of transgenic plant, and therefore continuing the clone and screening Δ 5 desaturases with higher Substratspezifitaet and catalytic activity is one of current research focuses.

Known Phaeodactylum tricornutum is rich in EPA, and high-content can reach more than 30% of total fatty acids, and Δ 5 desaturases should have high enzyme as EPA synthetic key enzyme and live.But the Substratspezifitaet of Δ 5 delta 8 desaturase genes that before obtain from this algae does not all include high (Domergue et al., 2002) with enzymic activity.In addition, do not retrieve relevant patent at home and abroad in the Patent data storehouse about marine microalgae Δ 5 delta 8 desaturase genes yet.

Summary of the invention

The object of the present invention is to provide a kind of Phaeodactylum tricornutum Δ 5 delta 8 desaturase genes, its proteins encoded and application.

Phaeodactylum tricornutum Δ 5 delta 8 desaturase genes of the present invention have the nucleotide sequence shown in the sequence table SEQ IDNO.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 sequence table 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 1431bp of Δ 5 delta 8 desaturase genes of the present invention, with the nucleotide sequence consistence (identity) of article one Δ 5 delta 8 desaturase genes (GenBank Accession No.AY082392) be 52%, 476 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 Phaeodactylum tricornutum is a template, utilize the primer shown in sequence table SEQ ID NO.3 and 4 to increase, the condition of amplification is: 94 ℃ of pre-sex change 3min, with 94 ℃ of 30s, 63 ℃ 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 5 desaturases of the present invention have the peculiar N terminal cell of front end desaturase pigment b5 structural domain and 3 Histidines bunch (176-180,215-219,413-417), and the 1st Histidine (H) of the 3rd Histidine bunch substituted by glutamine (Q).

Δ 5 desaturases of the present invention catalysis ETA (20:4 Δ 8,11,14,17) effectively generate EPA (20:5 Δ 5,8,11,14,17), and the Substratspezifitaet height, the enzymic activity height.

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 (Oleasp.), safflower (Carthamus sp.), cocoa (Theobroma cacca) and peanut (Arachis sp.) and seed, express this gene and Δ 6 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: pYES2 plasmid figure;

Fig. 2: Δ 5 desaturase catalysis lipid acid ETA generate the gas chromatographic analysis result of EPA.

Embodiment

Following embodiment is used for further specifying of the present invention, but is not used for limiting the scope of the invention.

The acquisition of embodiment 1 Δ 5 delta 8 desaturase genes

Concrete steps comprise:

(1) cultivation of Phaeodactylum tricornutum

1) 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).

2) preparation f/2 mother liquor (seeing Figure of description 3) and sterilization.

3) 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 table 1 Phaeodactylum tricornutum

Substratum	Salinity/‰	Temperature/℃	Initial pH	Illumination/μ mols -1m -2
					f/2	31	20±1	8.1	70(12h:12h)

The preparation of f/2 mother liquor

1. fundamental element:

NaNO ₃78.4g+1L DDW (distilled water);

NaSiO ₃·9H ₂O 10g+1L?DDW

NaH ₂PO ₄ 4.4g+1L?DDW

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

2. micro-:

ZnSO ₄·4H ₂O 2.3g

MnCl ₂·4H ₂O 17.8g

CuSO ₄·5H ₂O 1g

Na ₂MoO ₄·2H ₂O 0.73g

CoCl ₂·6H ₂O 1.2g

Na ₂EDTA 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 Na ₂EDTA, 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

FeC ₆H ₅O ₇·5H ₂O 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

V _B12 0.5mg

V _B1 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 tawny during little Nitzschia closterium minutissima normal growth, but the frond long period be suspended in the substratum, algae liquid is the cloud water body when rocking.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) degenerate pcr obtains partial sequence

The protein sequence of search Δ 5 fatty acid desaturases in GenBank has selected wherein that the sequence of 8 kinds of biologies compares, and according to comparative result, selects conservative relatively site design pair of degenerate primers:

Sense：5’-CA(C/T)CA(C/T)GCITA(C/T)ACIAA-3’HHAYTN

Antisense：5’-GG(A/G)AAIA(A/G)(A/G)TG(A/G)TG(T/C)TC-3’EHHLFP

Wherein, I represents Trophicardyl (inosine).

Carry out the condition optimizing of degenerate pcr earlier, the PCR reaction system and the reaction conditions of Que Dinging is as follows at last:

Sterilization DDW:6.75 μ L

10×PCR?buffer：1uL

dNTP(2.5mM/each)：0.8μL

genome?DNA：1μL(2ng/μL)

Forward?primer：0.2μL(50pmoL/μL)

Reverse?primer：0.2μL(50pmoL/μL)

Taq?DNA?poLymerase：0.05μL(5U/μL)

Amount to: 10 μ L

3 control tube are set simultaneously, are respectively and only add forward primer, only add the contrast of reverse primer, blank water, total reaction system still is 10 μ L.

Degenerate pcr product electrophoresis has reclaimed the purpose band of about 600bp, is cloned on the pMD18T-vector (TaKaRa) according to ordinary method, is checked order by Shanghai Ying Jun Bioisystech Co., Ltd.

(3) inverse PCR obtains full length gene

Sequencing result according to the degenerate pcr product designs a pair of special primer, and the position is in degenerated primer, and forward primer is at 3 ' end of known array and to its downstream amplification, and reverse primer is at 5 ' end and toward the upstream amplification.

Forward：5’-AGTGACAACGCGAACACCACC-3’

Reverse：5’-ATGCGATGCGGGATACTCTCG-3’

Simultaneously, analyze the restriction map (Restriction Map) of degenerate pcr product, be chosen in 6 kinds of restriction enzymes that do not have restriction enzyme site in this section sequence, enzyme is cut the genomic dna of Phaeodactylum tricornutum.Reaction system is as follows:

Sterilization DDW:42 μ L

genome?DNA：1μL(≈1μg/μL)

10×buffer：5μL H M H K H M

Restriction endonuclease: 2 μ L BglII BssH II EcoR I Hpa I Nde I Nhe I

37 ℃ of TotaL:50uL, enzyme is cut and is spent the night

Be pcr template.

Inverse PCR reaction system and reaction conditions are as follows:

Sterilization DDW:2.35 μ L

2×GC?I?buffer：5μL

dNTP(2.0mM/each)：1μL

Enzyme is cut from connecting product: 1 μ L (2ng/ μ L)

Forward?primer：0.3μL(10pmoL/μL)

Reverse?primer：0.3μL(10pmoL/μL)

LaTaq?DNA?poLymerase：0.05μL(5U/μL)

Amount to: 10 μ L

Found that cutting from connecting product with Hpa I enzyme is the band that template can obtain an about 2500bp of length, and be single band.The PCR product reclaims, and is cloned on the pMD18T-vector (TaKaRa) order-checking according to ordinary method.

(4) RT-PCR obtains the mRNA sequence

The full length sequence that is obtained by inverse PCR designs a pair of special primer:

Xho?I?5’end：5’-CCG CTCGAGATGTGCGTGACGACGCCGGTAACG-3’

Xba?I?3’end：5’-TGC TCTAGATTACTCCAATTTGGTAACTGCGTCCG-3’

RT reaction system and reaction conditions are as follows:

1) the total RNA+DEPC water of about 2 μ g (totally 22 μ L), 70 ℃, 5min places on ice immediately;

2) 5 * buffer, 10 μ L+dNTP (10mM each), 10 μ L+oligo dT (2.5mM), 5 μ L+HPRI, 1 μ L+AMV, 2 μ L, 42 ℃, 60min, 95 ℃, 5min, 4 ℃ of preservations.PCR reaction system and reaction conditions are as follows:

Sterilization DDW:5.4 μ L

10×buffer：1μL

dNTP(2.0mM/each)：1μL

Enzyme is cut from connecting product: 1 μ L (2ng/ μ L)

Xho?I?5’end：0.3μL(10pmoL/μL)

Xba?I?3’end：0.3μL(10pmoL/μL)

LaTaq?DNA?poLymerase：0.05μL(5U/μL)

Amount to: 10 μ L

The PCR product is cloned into the pBS carrier according to ordinary method, order-checking.Obtain Δ 5 delta 8 desaturase genes, its nucleotide sequence is shown in SEQ ID NO.1.

(5) according to above-mentioned sequencing result, the 2nd Δ 5 fatty acid desaturase mrna lengths from Phaeodactylum tricornutum are 1431bp, 477 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 pYD5-2 and the conversion of yeast saccharomyces cerevisiae

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

Test the long 5856bp of used yeast saccharomyces cerevisiae expression vector pYES2 (Invitrogen) (Fig. 1), 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 (galactose) then can inducible transcription.In addition, cell can also be grown in the substratum of raffinose (raffinose) as carbon source, raffinose is neither induced and is not also suppressed to transcribe, adding semi-lactosi under the situation of its existence in substratum equally can inducible transcription, 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 trp1-289ura3-52/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 (Uracil).

Other details are referring to the pYES2 specification sheets (Catalog no.V825-20) of Invitrogen.

The structure of 2 expression vector pYD5-2 and the screening of positive colony

1) extracts total RNA of frustule and do reverse transcription;

2) gene clone

Analyze the restriction map of this gene mRNA sequence, the MCS with reference to plasmid pYES2 designs a pair of special primer simultaneously:

D5-2NH：5-GCGAAGCTTACCATGGCTTGCGTGACGACGCCGGTAAC-3’

D5-2CE：5’-GCGGAATTCTTACTCCAATTTGGTAATTGCGTCCGTATTTC-3’

Wherein forward primer has Hind III restriction enzyme site, and recommends according to the plasmid specification sheets, and " ACCATGG " helps the expression of foreign gene in yeast cell, and in order not make sequence generation frameshit, the ATG back has added GCT, is translated as L-Ala.Reverse primer has the EcoRI restriction enzyme site.The PCR reaction system is as follows:

Sterilization DDW:27.75 μ L

5×PS?buffer：10μL

dNTP(2.5mM/each)：4μL

cDNA：5μL

D5-2NH：1.5μL(10pmol/μL)

D5-2CE：1.5μL(10pmol/μL)

Prime STAR high-fidelity enzyme: 0.25 μ L (2.5U/ μ L)

Amount to: 50 μ L

3) the PCR product reclaims, and uses Hind III and EcoR I double digestion simultaneously with pYES2;

4) reclaim enzyme and cut product, connect, transform.Choose mono-clonal and shake bacterium upgrading grain, PCR, double digestion identify that the positive clone of preliminary judgement send company's order-checking.The correct clone that checks order shakes bacterium upgrading grain in a large number, and-20 ℃ of preservations go to yeast conversion.

The preparation of 3 yeast saccharomyces cerevisiae competent cells and conversion

1) INVSc1 rules on the YPD flat board, 30 ℃ of cultivations;

2) single bacterium colony of the about 2mm of picking diameter is in 5mL liquid YPD substratum, and 30 ℃ are shaken and spend the night;

3) get an amount of bacterium liquid and insert the fresh YPD of 50mL, to OD ₆₀₀About 0.2,30 ℃, 250rpm to OD ₆₀₀About 1.0;

4) room temperature, 1500rpm, 3min, 1 * TE (10mM Tris, pH 7.5; 1mM EDTA) wash, 1mL 1 * TE is resuspended, goes to the EP pipe;

5) 8000rpm, 5s abandons supernatant, and precipitation is resuspended in 1mL 1 * TE/1 * LiAc (100mM, pH 7.5);

6) the centrifugal supernatant that goes, precipitation is resuspended in 250 μ L, 1 * TE/1 * LiAc, and room temperature is placed 10min;

7) get 50 μ L competent cells, to wherein adding 240 μ L PEG3350,36 μ L10 * LiAc (1M, pH 7.5), 25 μ L sex change salmon sperm DNAs (2mg/mL), 50 μ L pYD5-2 or pYES2 empty plasmid contrast (1 μ g) successively, the whirlpool hangs mixing;

8) 30 ℃ of shaking table jog 30-60min;

9) add 40 μ L DMSO, mixing gently, 42 ℃ of thermal shock 15min;

10) 6000rpm, 15s thoroughly removes supernatant, and 1mL 1 * TE washes;

11) 6000rpm, 15s thoroughly removes supernatant, and 05mL 1 * TE is resuspended, coating SC-U (2%glucose) selectivity flat board, 30 ℃ left standstill 3-4 days, treated the dull and stereotyped clone that upward grown.

12) picking yeast list bacterium colony is cultivated, and extracts zymic DNA, is PCR with special primer D5-2NH and D5-2CE, and the contrast of changeing empty plasmid is set.It is the positive colony that has changed the D5-2 gene over to that PCR can access segmental this yeast that shows of purpose, is used to do express test.

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%raffinose) liquid nutrient medium, 30 ℃ are shaken and spend the night;

3) it is centrifugal to get an amount of bacterium liquid, adds 3.6ml SC-U (2%galactose), to initial OD ₆₀₀About 0.2, add 400 μ L 10%NP-40 (final concentration 1%), add lipid acid substrate (20:4n3) simultaneously to final concentration 50 μ M, induce 72h for 20 ℃.

2 lipid acid extract and analyze

2.1 lipid acid extracts

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

2) liquid nitrogen grinding changes the dry powder after grinding over to the EP pipe, adds 350 μ L chloroforms: and methyl alcohol (2: 1, v/v), vortex;

3) supersound process, super 3s stops 3s, 22% power, 3min notices that the EP pipe places frozen water;

4) every pipe adds the granulated glass sphere of a little pickling in the new EP pipe, changes the liquid of supersound process over to new pipe, former effective 350 μ L chloroforms: and methyl alcohol (2: 1, v/v) to wash, washing lotion changes new pipe equally over to, and vortex 1min stops 1min, repeats 3-4 time;

5) cooled with liquid nitrogen 5min changes 4 ℃ over to, cracking 2h;

6) 4 ℃, 12000rpm, 5min, supernatant move into new pipe;

7) add 300 μ L 0.9%KCl (w/v), vortex 1min stops 1min, repeats 3-4 time;

8) 4 ℃, 12000rpm, 5min, lower floor's chloroform layer move into new EP pipe, and former effective 500 μ L chloroforms are washed the KCl layer one time again, and same vortex 3-4 time is centrifugal, sucking-off lower floor chloroform and original merging;

9) chloroform is removed in vacuum-drying;

2.2 methyl esterification of fatty acid

1) it is molten that the lipid acid of Ti Quing adds 300 μ L normal hexanes weight, adds 750 μ L HCl-CH ₃OH (1N), the vortex mixing;

2) 85 ℃, the 2h esterification;

3) change-20 ℃ over to, the 20min cooling;

4) add 350 μ L 0.9%KCl, vortex, static layering is carefully got the upper strata normal hexane, changes new EP pipe over to, can add the extracting one time again of an amount of normal hexane as needs in former pipe, merges normal hexane;

5) vacuum-drying, heavy molten before last sample is analyzed with 50 μ L normal hexanes.

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

1) instrument: Agilent (Agilent) 6890; Chromatographic column model: Varian CP7638;

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

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

Temperature programming: 100 ℃ of (2min) → 10 ℃/min rises to 180 ℃ of (0min) → 4 ℃/min and rises to 260 ℃ (15min);

Detector: flame ionization ditector (FID);

2) the various fatty acid methyl esters of producing with Sigma company are standard substance;

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

4) analysis software: HPCHEM chromatographic working station.

3 test-results

Gas chromatographic analysis shows, the lipid acid substrate 20:4n3 that external source adds can be converted into 20:5n3 (EPA) after having changed the yeast abduction delivering of pYD5-2 over to, the albumen that institute's transgenes encoding is described has the function of Δ 5 fatty acid desaturases really, is defined as the 2nd Δ 5 fatty acid desaturases of Phaeodactylum tricornutum.

The stratographic analysis result as shown in Figure 2, A is for adding behind the substrate without the inductive contrast, B is for adding the result of semi-lactosi abduction delivering.

Lipid acid is formed

Lipid acid	% (accounting for total fatty acids)
		14:0 16:0 16:1Δ9 18:0 18:1Δ9 20:4Δ8，11，14，17 20:5Δ5，8，11，14，17	29.7 13.8 26.6 4.5 19.8 1.1 2.9

Add semi-lactosi and induce and 70% external source substrate 20:4 Δ 8,11,14,17 can be converted into EPA, and contrast the EPA that arrives that does not detect.

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Sequence table

Claims (8)

1. marine microalgae Δ 5 fatty acid desaturases, its aminoacid sequence is shown in SEQ IDNO.2.

2. the gene of coding claim 1 described marine microalgae Δ 5 fatty acid desaturases.

3. gene as claimed in claim 2, its nucleotide sequence is shown in sequence table SEQ IDNO.1.

4. the expression vector that contains claim 2 or 3 described sequences.

5. the host cell that contains the described expression vector of claim 4.

6. host cell as claimed in claim 5, it is the oilseed plant cell.

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

8. the application of the described enzyme of claim 1 in preparation DHA.