CN100552029C - Pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence and application thereof - Google Patents

Abstract

The present invention relates to a kind of omega 3-fatty acid dehydrogenase promoter sequence that from pichia spp, is separated to and application thereof.The present invention includes and have the nucleotide sequence shown in the SEQ ID NO:1.The present invention includes and utilize this promoter sequence to change the microbiological genetic engineering method of microorganism cells protein component, in particular, be connected allos or homologous gene in this promotor downstream, and be building up to microbial expression vector, transform host microorganism, in its cell, express target protein, realize quality-improving or pharmaceutical usage.The omega 3-fatty acid dehydrogenase gene promoter that the present invention clones pichia spp has the major application prospect to the research of all transgenic microorganisms from now on.

Description

Pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence and application thereof

Technical field

The invention belongs to biological technical field, particularly a kind of isolating omega 3-fatty acid dehydrogenase promoter sequence and application thereof from pichia spp.From the total DNA of pichia spp, clone this omega 3-fatty acid dehydrogenase promoter, and connect, transform different microorganisms, make its transgenic microorganism express the methods and applications of its downstream gene with different expression vector.

Background technology

PUFAs (polyunsaturated fatty acids) is to be present in a variety of forms in the cell, as cytolemma, storage fat, triglyceride, sphingolipid and lipoprotein etc., exercises different biological functions in cell.PUFAs mainly contains two big classes, n-6 and n-3, and they are that (Linoleic acid, LA is expressed as 18:2 with linolic acid respectively ^9,12N-6) and alpha-linolenic acid (α-Linolenic acid, ALA is expressed as the 18:3 Δ ^9,12,15N-3) be the substrate synthetic.Two class unsaturated fatty acidss are pathways metabolism difference not only in vivo, and different biological activitys is arranged, and for example in n-6 class PUFA: LA is the important composition composition of ceramide; AA is the precursor of eicosanoid, and eicosanoid comprises function hormones such as leukotrienes, prostaglandin(PG) and thromboxane, and these hormones participate in some physiology and pathologic process, as it is initial to give a birth, platelet aggregation, the ionogen of kidney is regulated, the activation of blastocyte implantation and immunocyte; N-6 PUFA also may be in the effect of serving as the second messenger aspect the intercellular signal conduction.And at n-3 class PUFA except providing for metabolic process energy and the carbon atom, EPA and DHA also can be used as the precursor of eicosanoid, but compare with AA deutero-eicosanoid, just in the very high tissue of EPA and DHA content, could form a considerable amount of these hormonal substances, so may just play some antiphlogistic effects; DHA is playing very important function aspect the cell membrane function of keeping retina and nervous tissue.If lack n-3 PUFAs in retina and the nervous tissue then can be by the substituting of 22:5n-6 compensatory, such minor alteration can be enough to cause the forfeiture of memory and the damage of eyesight.

In most of fungi and plant materials, n-6 class unsaturated fatty acids all is through ω ³The catalytic dehydrogenation of-fatty acid dehydrogenase generates n-3 class unsaturated fatty acids, so ω ³-fatty acid dehydrogenase is the rate-limiting enzyme of n-3 class high unsaturated fatty acid building-up process, therefore be subjected to the adjusting of a plurality of levels, for example substrate or product horizontal adjustment, temperature regulation, the cell regulate and control factor is regulated or the like, and these adjustings all are to realize by direct or indirect interacting with its upstream promoter.Though in multiple organism, be cloned into ω at present ³-fatty acid dehydrogenase gene sequence, but the clone and the analysis of its promoter sequence do not appeared in the newspapers as yet.

Summary of the invention

The purpose of this invention is to provide a kind of isolating omega 3-fatty acid dehydrogenase promoter sequence and application thereof from pichia spp.The invention reside in and make its expression of exogenous gene in the transgenic microorganism, the content of alpha-linolenic acid adjusting in the expression regulation of omega 3-fatty acid dehydrogenase and the thalline in the pichia spp.Therefore, the present invention produces medicinal alpha-linolenic acid to improvement transgenic microorganism quality and high unsaturated fatty acid is very useful.

The invention provides isolating omega 3-fatty acid dehydrogenase promoter nucleotide sequence from pichia spp, specifically is pichia pastoris omega 3-fatty acid dehydrogenase promoter nucleotide sequence and application thereof.This promotor downstream is connected other foreign gene, and connects, be transformed in the microorganism, express the methods and applications of its downstream gene with different expression vector.

The purpose of this invention is to provide and contain this promotor nucleotide sequence and be connected the expression vector of constructing function expression with other foreign gene.

Another object of the present invention provides and contains expression vector transformed host cell or host's spore and the offspring thereof that this promotor nucleotide sequence or this promotor nucleotide sequence are connected with other foreign gene.

Another object of the present invention provides a kind of usefulness and contains expression vector transformed host cell or host's spore and the progeny cell thereof that this promotor nucleotide sequence or this promotor nucleotide sequence are connected with other foreign gene, and carries out the transgenic microorganism quality-improving with this and produce medicinal alpha-linolenic acid and high unsaturated fatty acid etc.

Pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence provided by the invention comprises:

(1) has the nucleotide sequence shown in the SEQ ID NO:1; Or

(2) with (1) described nucleotide sequence complementary nucleotide sequence; Or

(3) nucleotide sequence that has the basic sequence homology with the nucleotide sequence of (1) or (2); Or

(4) be the nucleotide sequence of analogue of the nucleotide sequence of (1), (2) or (3); Or

(5) with the nucleotide sequence of (1), (2), (3) or (4), the nucleotide sequence of under hybridization conditions, hybridizing.

A second aspect of the present invention provides and has contained above-mentioned nucleotide sequence and the constructed recombinant expression vector of plasmid vector.As expression vector pYEPppw3P.

The invention provides a kind of genetically engineered host cell, it is to be selected from: the 1) host cell that transforms or transduce with described nucleotide sequence; 2) host cell that transforms or transduce with described recombinant expression vector.Described host cell is that offspring, the protein of microbial host cell is formed the microorganism cells that has changed.Described microorganism is intestinal bacteria or yeast saccharomyces cerevisiae.

Described host cell is a brewing yeast cell.

The invention provides a kind of usefulness contains expression vector transformed host cells that this promotor nucleotide sequence or this promotor nucleotide sequence be connected with other foreign gene and progeny cell thereof and carries out transgenic microorganism protein and form improvement and produce medicinal alpha-linolenic acid and high unsaturated fatty acid etc.

The method of expressing omega 3-fatty acid dehydrogenase promoter in microorganism cells of the present invention comprises the steps:

1) the expression vector carrier that will contain above-mentioned nucleotide sequence imports microorganism cells.

2) make described microorganism cells growth form somatic cells.

The present invention can be used for producing human food prods, animal-feed, makeup or medicine.

The separation method of pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence of the present invention comprises the steps:

1) extracts the total DNA of pichia spp, with genome SalI single endonuclease digestion.

2) with Mortierella isabellina (Mortierella isabellina) Δ 6-fatty acid dehydrogenase open reading frame double digestion (BamHI﹠amp; SalI) gained fragment (663bp-1090bp, SEQ ID NO:2) is as joint, enzyme cut the back genome spend the night with 1: 3 in molar ratio ratio of joint and be connected.

3) design and synthesize following three primers:

Primer 1:GAACTCCTCA GCGGTGTATG GAACAAAGAC

Primer 2: ATGACCTGTT GCCTTATGAT GC

Primer 3:GGCGATTGT GTTCTCGCTC

4) be template to connect product, carry out the PCR reaction:

The reactive component add-on

Damping fluid (10 *)

(contain 20mmol/L MgCl ₂) 5 μ l

dNTP(2.5mmol/L) 2μl

Primer 1 (1 μ mol/L) 2 μ l

Primer 2 (1 μ mol/L) 2 μ l

Taq(5u/μl) 0.5μl

H ₂O 36.5μl

Template DNA (0.1 μ g/ μ l) 1 μ l

Cumulative volume 50 μ l

Amplification condition: 94 ℃ of 5min; 94 ℃ of 30s, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; 72 ℃ of 10min.

5) with 100 times of dilutions of PCR product as template, with primer 1,3, as primer, as above system, as above condition is carried out PCR.

6) reclaim the PCR fragment, subclone is to sequencing vector; Be transformed into competent escherichia coli cell again, containing penbritin, final concentration is an overnight incubation on LB (Luria-Bertani) solid medium of 50-100 μ g/ml; The white colony of growing on the picking flat board inserts overnight incubation in the LB liquid nutrient medium that contains penbritin, and centrifugal collection thalline is by the alkaline lysis method of extracting plasmid, through NcoI and evaluation of SacI double digestion and pcr amplification evaluation positive colony.

7) the positive colony plasmid is checked order the conclusive evidence promoter fragment.

Nucleotide sequence described in the present invention can be that wherein one or more Nucleotide replace, the nucleotide sequence of disappearance, insertion, inversion, i.e. the artificial mutant of original separation sequence, and it also has its promoter function.It can also be the fusion sequence of described nucleotide sequence and other promoter sequence.

Other foreign gene described in the present invention is meant any one section nucleotide sequence, and has coding certain protein or other active substance function, comprises RNA or dna sequence dna, and this sequence does not combine with the omega 3-fatty acid dehydrogenase promoter normal circumstances.This nucleotide sequence comprises the heterologous nucleotide sequence that the microbe species that is different from promotor obtains, and the homologous nucleotide sequence that obtains from the microbe species that is same as promotor, but the promotor of these sequences and wild-type (non-transgenic) microorganism is irrelevant.

Any carrier that expression vector described in the present invention is meant is well known in the prior art, can express in microorganism, for example pYES2.0, pYEP356 etc.

Any method for transformation that conversion described in the present invention is meant is well known in the prior art, can import foreign gene microorganism cells or microbial spore is as cell electricimpulse conversion method etc.

Host cell described in the present invention or and progeny cell is meant all microorganism cellss or microbial spore or by the microbe of these cells or sporogenesis.

Term " nucleotide sequence " refers to contain naturally occurring base, the sequence of the Nucleotide of the key of (side chain) or nucleoside monomers between sugar and the sugar.This sequence also comprises and contains the monomer that the non-natural of bringing into play identity function exists or sequence modification or that replace of its part.Be meant oligonucleotide, Nucleotide or polynucleotide and fragment thereof or part, also can also refer to genome or synthetic DNA, they can be strand or two strands, and representative has justice.

Term " omega 3-fatty acid dehydrogenase promoter " is meant that the gene of expressing is being with or without in the microorganism cells of primary expression and preferentially expresses under promotor control.

Term " sequence with basic sequence homology " is meant that having the nucleotide sequence shown in the SEQ ID NO:1 or (2) with (1) compares with the sequence among (1) has the nucleotide sequence complementary nucleotide sequence shown in the SEQ ID NO:1 those nucleotide sequences slight or inessential sequence variation are arranged, and these sequences play a role in essentially identical mode and can drive the expression of its downstream gene.These variations may be because partial sudden change or structural modification.

Term " sequence of hybridization " be meant can be under stringent hybridization condition with described nucleotide sequence (1), (2), (3) or (4) among the nucleotide sequence of sequence hybridization." stringent hybridization condition " is meant well known by persons skilled in the art, perhaps can find in the general scheme in " molecular cloning experiment guide " (J. Sa nurse Brooker, E.F. not Ritchie, T. Manny A Disi write Science Press, second edition, 1993).

The present invention clone's Pichia pastoris omega 3-fatty acid dehydrogenase gene promoter is the Pichia pastoris omega 3-fatty acid dehydrogenase gene promoter of being cloned into first, and is the active functional verification of omega 3-fatty acid dehydrogenase promoter of this promotor.The invention reside in and make its expression of exogenous gene in the transgenic microorganism, avoided foreign gene in other position continuous expression adverse effect.Therefore, that transgenic microorganism is produced pharmaceutical protein is very useful in the present invention.The research and development of this promotor, to transgenic plant from now on, transgenic microorganism has the major application prospect.

Description of drawings

Fig. 1: yeast saccharomyces cerevisiae expression vector pYEPpp3 synoptic diagram.

Fig. 2: yeast saccharomyces cerevisiae recombinant expression vector pYEPppw3P synoptic diagram.

The gas chromatogram of Fig. 3 A, demonstration alpha-linolenic acid methyl ester standard substance.

Fig. 3 B, contain the gas chromatogram of the transgenic yeast of pYEPppw3 carrier.

Fig. 3 C, contain the gas chromatogram of the transgenic yeast of recombinant plasmid pYEPppw3P.

Embodiment

The separation of embodiment 1. Pichia pastoris omega 3-fatty acid dehydrogenase gene promoter sequences (ppw3P)

Extract the total DNA of pichia spp, with genome SalI single endonuclease digestion.With Mortierella isabellina (Mortierellaisabellina) Δ 6-fatty acid dehydrogenase open reading frame double digestion (BamHI﹠amp; SaII) gained fragment (663bp-1090bp, SEQ ID NO:2) is as joint, enzyme cut the back genome spend the night with 1: 3 in molar ratio ratio of joint and be connected.Design and synthesize following three primers:

Primer 1:GAACTCCTCA GCGGTGTATG GAACAAAGAC

Primer 2: ATGACCTGTT GCCTTATGAT GC

Primer 3:GGCGATTGT GTTCTCGCTC

To connect product is template, carries out the PCR reaction:

The reactive component add-on

Damping fluid (10 *)

(contain 20mmol/L MgCl ₂) 5 μ l

dNTP(2.5mmol/L) 2μl

Primer 1 (1 μ mol/L) 2 μ l

Primer 2 (1 μ mol/L) 2 μ l

Taq(5u/μl) 0.5μl

H ₂O 36.5μl

Template DNA (0.1 μ g/ μ l) 1 μ l

Cumulative volume 50 μ l

Amplification condition: 94 ℃ of 5min; 94 ℃ of 30s, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; 72 ℃ of 10min.

With 100 times of dilutions of PCR product as template, with primer 1,3, as primer, as above system, as above condition is carried out PCR.Reclaim the PCR fragment, subclone is to sequencing vector; Be transformed into competent escherichia coli cell again, containing penbritin, final concentration is an overnight incubation on the LB solid medium of 50-100 μ g/ml; The white colony of growing on the picking flat board inserts overnight incubation in the LB liquid nutrient medium that contains penbritin, and centrifugal collection thalline is by the alkaline lysis method of extracting plasmid, through NcoI and evaluation of SacI double digestion and pcr amplification evaluation positive colony.The positive colony plasmid is checked order, obtain containing potential promoter sequence (SEQ ID NO:1, the upstream sequence of omega 3-fatty acid dehydrogenase 392-779) (SEQ ID NO:1).

Embodiment 2: the structure of yeast saccharomyces cerevisiae recombinant expression vector

According to SEQ ID NO:1, sequence shown in the 392-779, design gene specific amplimer (primer 5 and 6) and separate its potential promoter sequence:

Primer 4:5-GGCAAGCTTATGTCAAAAGTCACTGTTTCGGG-3`;

Primer 5:5`-GCCTCTAGATTAGGTATCC TTAGG-3`;

Primer 6:5`-GGCGGTACCGCCGAAGAAAGTAGAAGAGAAG-3`

Primer 4 and 5 5` end black matrix contain HindIII and XbaI enzyme cutting site respectively, are used to separate ω <sup>3</sup>The open reading frame of-fatty acid dehydrogenase (ppw3).Primer 6 and 5 5` end black matrix contain KpnI and XbaI enzyme cutting site respectively, are used to separate ω ³The open reading frame of-fatty acid dehydrogenase and potential promoter sequence (ppw3p) thereof.Used amplification condition and reactive component are the same, get 50 μ l PCR products then respectively and 1 μ l pYEP356 carries out double digestion respectively, reclaim enzyme and cut big fragment, and with the T4 ligase enzyme in 4 degree refrigerator overnight connections.Connect product transformed into escherichia coli DH5 α, extract and the PCR screening positive clone by plasmid, and the evaluation of checking order.Plasmid construction the results are shown in accompanying drawing 2 and 3, the constructed open reading frame expression plasmid of yeast called after pYEPppw3 that contains the omega 3-fatty acid dehydrogenase gene.This plasmid is to be made up by the PCR product of pYEP356 and primer 4 and primer 5 to form.Plasmid and respectively behind HindIII and XbaI double digestion, electrophoresis reclaims purifying, connects through T4DNA Ligase, connects product transformed into escherichia coli DH5 α, the Screening and Identification matter example that goes out to recombinate, called after pYEPppw3.The expression plasmid of yeast called after pYEPppw3p of constructed open reading frame that contains the omega 3-fatty acid dehydrogenase gene and potential promoter sequence thereof.This plasmid is to be made up by the PCR product of pYEP356 and primer 6 and primer 5 to form.Plasmid and respectively behind KpnI and XbaI double digestion, electrophoresis reclaims purifying, connects through T4DNA Ligase, connects product transformed into escherichia coli DH5 α, and Screening and Identification goes out recombinant plasmid, called after pYEPppw3p.

Embodiment 3: the recombinant plasmid yeast saccharomyces cerevisiae of electric shock mediation transforms

Wine brewing yeast strain INVScl streak inoculation from the glycerine pipe is cultivated 48h for 30 ℃ and activated on the YEPD flat board; Picking list bacterium colony is in 5ml YEPD liquid nutrient medium, and 30 ℃, 250rpm shaken overnight are cultivated; Detect the OD of bacterium liquid ₆₀₀Value is got an amount of bacterium liquid and is diluted in the 50ml YEPD liquid nutrient medium, makes OD ₆₀₀=0.4, continue to cultivate 2-4h; The 2500rpm centrifugation cell removes supernatant, adds the sterilized water centrifuge washing twice that 50ml shifts to an earlier date ice bath, twice of the 1mol/L sorbyl alcohol centrifuge washing of ice bath; 2ml 1mol/L sorbyl alcohol re-suspended cell re-suspended cell, and with the packing of 100 μ l/ pipe, as for using as early as possible on ice.In 100 μ l competence yeast cell, add the soft mixing of 10 μ g recombinant expression plasmids; MicroPulser is set to " ScaI " retaining, parameter is: Voltage 2.0kV, Number of pulsesl; Plasmid and yeast cell mixed solution are changed in the 0.2cm electric shock cup that shifts to an earlier date ice bath, make liquid feed flow to the pipe end, as for shocking by electricity on the electric conversion instrument; Take off the electric shock cup; Add the 1mol/L sorbyl alcohol of 1ml ice bath immediately and coat on SC-Ura (no uridylic) the selectivity solid medium (containing 2% glucose), put 30 ℃ and cultivate 48h or occur until transformant

Embodiment 5: the abduction delivering of Yeast engineering bacteria

The positive that picking SC-Ura (no uridylic) selects to occur on the culture medium flat plate transforms, be inoculated in 15ml SC-Ura and select substratum (containing 2% glucose), 28 ℃ are shaken the bacterium incubated overnight, add the 100ml SC-Ura substratum that contains 2% glucose with 5% inoculum size, 28 ℃ are continued to cultivate 72h; 2500rpm collects thalline, uses deionized water wash three times, and 50 ℃ of oven dry grind, and get the KOH-CH that 100mg adds 5ml 5% ₃OH solution, 70 ℃ of reaction 2-3h; Reaction finishes, and cool to room temperature is with the pH value to 2.0 of the hydrochloric acid conditioning solution of 6mol/L, adding 4ml 14%BF ₃-CH ₃OH, 70 ℃ of reaction 1.5h, synthesizing fatty acid methyl ester; Add saturated NaCl solution 10ml again, the concuss mixing, and transfer in the separating funnel, use 1: 4 chloroform of 8ml: hexane extracting twice, united extraction liquid; Add an amount of anhydrous Na ₂SO ₄Dry extraction liquid leaves standstill 1h, removes Na ₂SO ₄, the supernatant liquor that contains fatty acid methyl ester is dried up with nitrogen, return molten sample with the normal hexane of 200 μ L, the back is with the filtering with microporous membrane of 0.45mm.

Embodiment 6: the lipid acid gas chromatographic analysis

Undertaken by following condition:

Instrument is Tianjin, island GC-7A, pillar: fused-silica capillary column, 0.32 * 30m, solid support: Dienthyeneglycol succinate (Poly-diethylene glycol succinate, DEGS) plated film thing: polyimide.Carrier gas: N ₂, linear speed: 10cm/s.Splitting ratio: 100: 1, the vaporizer temperature: 250 ℃, column temperature: 180 ℃, tail blew: 50ml/min, detector: flame ionization ditector.The LA methyl esters of producing with Sigma company is standard substance, the sample of the methyl esterification of fatty acid of method for preparing, carries out GC and analyzes, and applied sample amount is 1 μ l; Analysis software: Anstar, the star color spectrum workstation of analysis.

Stratographic analysis the results are shown in accompanying drawing 4, and accompanying drawing 4 shows linolic acid methyl ester standard substances (4A), contain the transgenic yeast (4B) of pYEPppw3 carrier and contain the gas chromatogram of the transgenic yeast (4C) of recombinant plasmid pYEPppw3p.Compare by retention time and to identify each peak with known fatty acid methyl ester standard substance.Retention time is that the peak of 14.76min correspondence is the linolic acid that Pichia pastoris omega 3-fatty acid dehydrogenase catalysis oleic acid produces among Fig. 4 C, and does not have corresponding peak to occur among Fig. 4 B.Hence one can see that, the upstream sequence of isolating omega 3-fatty acid dehydrogenase have promoter activity.

Sequence list SEQUENCE LISTING

＜110〉Nankai University

＜120〉pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence and application thereof

<130>2006?07?21

<160>2

<170>Patentln?version?3.3

<210>1

<211>780

<212>DNA

＜213〉pichia spp (Pichia pastoris)

<400>1

gtcgacattg?tcaagcgaag?aagattacta?aacattttca?ctgagcatgt?agtcacactg 60

tcttgcaatg?cttatggatc?acacattatg?gacaaattat?ggcaattctc?catcaagttg 120

aatctcttca?aagacagaat?tgcactggct?ctgtttgaaa?acaaagacaa?aatcaaggac 180

agcatctatg?gaaaacttgt?ttggaagaac?tggtctatgg?agttatattg?ccgaagaatg 240

tatgactgga?aaaatcttat?aaaacagcag?gagttggagc?ttttccccga?tcatagagga 300

gctccaactc?tcttgaagaa?aaggccccta?gaagagccga?agaaagtaga?agagaagaag 360

gacaaaagta?gaggacgtag?aagatatcat?tagaagtaaa?ttaatatata?attgttatta 420

taattgtgct?tttttatagg?gtcctttgaa?gtaatgggcg?agctctattg?tacccaaaat 480

ctggggatga?accaaattac?acgtaaaacc?tatacaactt?ttaatttcta?tttccaacca 540

aaaatgtcat?atttgacggc?aacacgttca?agtcaccaca?agaaagtctc?ctagagtgta 600

atacaccttt?agtattttaa?taatataatg?gccaataata?aaagcctaat?acctatcact 660

aatctcacaa?cactgaggac?gtcgcttttc?ccgatatgga?aatgtcagag?atcccgcctc 720

agtttaaaat?gatgaaaaaa?aaaatatcca?atttgaaccc?accacccaac?caacctgaaa 780

<210>2

<211>428

<212>DNA

＜213〉pichia spp (Pichia pastoris)

<400>2

ggatcccgac?attgacactc?accctctgtt?gacgtggagt?gagcatgctt?tggagatgtt 60

ctcggacgtc?cctgacgagg?agctgacccg?catgtggtcg?cgcttcatgg?tccttaacca 120

gacctggttc?tactttccca?ttctctcgtt?tgcccgtctc?tcctggtgcc?tccagtccat 180

cctctttgtt?ctgcctaacg?gtcaggccca?caagccctct?ggagcccgtg?tgcccatttc 240

cttggtcgag?cagctgtctc?ttgccgtgca?ctggacctgg?tacctcgcca?ccatgttctt 300

gttcattaag?gaccccgtca?acatgatggt?gtactttttg?gtgtctcagg?ctgtttgcgg 360

taacctgttg?gcgattgtgt?tctcgctcaa?ccacaacggt?atgcctgtga?tctccaagga 420

ggaagccg 428

Claims (8)

1, a kind of isolating pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence is characterized in that it comprises:

(1) nucleotide sequence shown in the SEQ ID NO:1; Or

(2) with (1) described nucleotide sequence complementary nucleotide sequence.

2, a kind of recombinant expression vector is characterized in that it is by described promoter sequence of claim 1 and the constructed recombinant expression vector of plasmid vector.

3, a kind of genetically engineered host cell is characterized in that it is to be selected from: with the host cell of described recombinant expression vector conversion of claim 2 or transduction.

4,, it is characterized in that described host cell is a yeast saccharomyces cerevisiae according to the described host cell of claim 3.

5, a kind of method by the described omega 3-fatty acid dehydrogenase promoter expression of claim 1 downstream gene is characterized in that its step comprises:

1) the described recombinant expression vector of claim 2 is imported microorganism cells;

2) make described microorganism cells growth form the microorganism cells of expressing downstream gene.

6, in accordance with the method for claim 5, it is characterized in that described microorganism is intestinal bacteria or yeast saccharomyces cerevisiae.

7, the separation method of the described pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence of claim 1 is characterized in that it comprises the steps:

1) extracts the total DNA of pichia spp, with genome SalI single endonuclease digestion;

2) with Mortierella isabellina (Mortierella isabellina) Δ 6-fatty acid dehydrogenase open reading frame double digestion BamHI﹠amp; SalI gained fragment 663bp-1090bp, SEQ ID NO:2 be as joint, enzyme cut the back genome spend the night with 1: 3 in molar ratio ratio of joint and be connected;

3) design and synthesize following three primers:

Primer 1:GAACTCCTCA GCGGTGTATG GAACAAAGAC

Primer 2: ATGACCTGTT GCCTTATGAT GC

Primer 3:GGCGATTGT GTTCTCGCTC

4) be template to connect product, carry out the PCR reaction:

The reactive component add-on

Damping fluid 10 *

Contain 20mmol/L MgCl ₂5 μ l

dNTP2.5mmol/L 2μl

Primer 11 μ mol/L 2 μ l

Primer 21 μ mol/L 2 μ l

Taq?5u/μl 0.5μl

H ₂O 36.5μl

Template DNA 0.1 μ g/ μ l 1 μ l

Cumulative volume 50 μ l

Amplification condition: 94 ℃ of 5min; 94 ℃ of 30s, 56 ℃ of 1min, 72 ℃ of 2min, totally 30 circulations; 72 ℃ of 10min;

5) with 100 times of dilutions of PCR product as template, with primer 1,3, as primer, as above system, as above condition is carried out PCR;

6) reclaim the PCR fragment, subclone is to sequencing vector; Be transformed into competent escherichia coli cell again, containing penbritin, final concentration is an overnight incubation on the LB solid medium of 50-100 μ g/ml; The white colony of growing on the picking flat board inserts overnight incubation in the LB liquid nutrient medium that contains penbritin, and centrifugal collection thalline is by the alkaline lysis method of extracting plasmid, through NcoI and evaluation of SacI double digestion and pcr amplification evaluation positive colony;

7) the positive colony plasmid is checked order the conclusive evidence promoter fragment.

8, the described pichia pastoris omega 3-fatty acid dehydrogenase promoter sequence of claim 1 is used to produce human food prods, animal-feed, makeup or medicine.

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