CN105200060B - Incise the FAD gene promoters of edge green alga Δ 6 and its application - Google Patents
Incise the FAD gene promoters of edge green alga Δ 6 and its application Download PDFInfo
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Abstract
The present invention relates to edge green alga Δ 6FAD gene promoters and its application is incised, purposes, the recombinant vector containing the promoter sequence and the genetically engineered host cell for incising edge green alga Δ 6FAD gene promoter sequences, the promoter sequence in algae expression target gene is started are specifically provided.Connected by the way that obtained edge green alga Δ 6FAD gene promoters of incising will be cloned into fusion expression vector, electroporated method transformed saccharomyces cerevisiae BY4741, it is found that the promoter can drive the expression of target gene, and its efficiency is nearly equivalent to T7 promoters.The present invention creates condition for the isogenic expression of artificial adjustment algae desaturase.
Description
Technical field
The present invention relates to genetic engineering field, specifically, be related to one kind incise the FAD gene promoters of edge green alga Δ 6 and
It is applied.
Background technology
Arachidonic acid (arachidonic acid, ArA, 20:4 ω 6) be human nutrition a kind of essential fatty acid.No
Only there is many physiology and pharmacological activity, and be the main component of human brain membrane phospholipid.It plays the second letter in the cell
Make effect, participate in hematolymphiod regulation, cause vasodilation, participate in the regulation of the organ multiple functions such as liver, courage, be interior cyclic system
The prostaglandin and the precursor of Bai Jie triolefins to be played an important role in system and central nervous system.ArA or ripe breast milk one kind
Important component, eyesight and intelligence development to postpartum baby are all required.Therefore, FAO/WHO recommends to add in baby milk
Add ArA.Incise edge green alga (Myrmecia incisa) and be rich in substantial amounts of ArA, especially (such as phosphate starvation, nitrogen are hungry in stressful environmental
Starve) under, ArA highest contents can reach the 7% of frond dry weight, be the higher algae kind of current ArA contents, have potential exploitation profit
With value.
ArA is a kind of polyunsaturated fatty acid (PUFA).PUFA synthesis in organism mainly has two kinds of approach, i.e. ω -6
Approach and ω -3 approach.ArA is synthesized along ω -6 approach：I.e. linoleic acid is first in the fatty acid desaturase of Δ 6 (FAD)
Gamma-Linolenic acid is generated under desaturation, generation bishomo-γ-linolenic acid is extended by the fatty acid elongase of Δ 6 (FAE) afterwards
(dihomo- γ-linolenic acid, 20:3Δ8,11,14, DGLA), then ArA is generated by Δ 5FAD desaturation.Research
It has also been found that linoleic acid can generate alpha-linolenic acid (ALA) in the presence of Δ 15FAD enters ω -3 approach, then, ALA can be in Δ 6
The lower desaturation of FAD effects is parinaric acid (stearidonic acid, SDA, 18:4Δ6,9,12,15), and pass through aliphatic acid
Desaturation acts on extension, further produces eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).Tocher and
Harvie research indicates that the first step catalytic dehydrogenating reaction of ω -6 and ω -3 route of synthesis is rate-limiting step, in ω -6 approach
In, the step for be the catalytic action by the FAD of Δ 6.By making the analysis of substrate Preference, hair to incising the FAD of edge green alga Δ 6
The existing FAD of Δ 6 can not only make LA become GLA, and ALA can also be catalyzed into SDA, but the efficiency of product is not high, so as to also say
Understand the FAD of Δ 6 speed limit effect.Obviously, the more big generation for being more advantageous to ArA of the transcription amount of the FAD genes of Δ 6.
It is well known that the transcription of gene is typically controlled by the promoter of the upstream region of gene 5 '-flanking sequence.Promoter sequence
Usually contain controlling element, the transcription of the gene is produced response to the environment of change, so as to show differential expression.Cause
This, in order to which further investigated incises the mechanism that edge green alga accumulates ArA under the adverse environmental factors such as nitrogen, it is necessary to know the algae Δ 6 FAD
The promoter and its function of gene.
The content of the invention
The purpose of the present invention is to be directed to deficiency of the prior art, there is provided a kind of polynucleotides of separation.
Another purpose of the present invention is to provide the purposes of described polynucleotides.
Another purpose of the present invention is to provide a kind of recombinant vector.
Fourth object of the present invention is to provide a kind of genetically engineered host cell.
To realize above-mentioned first purpose, the present invention adopts the technical scheme that：
A kind of polynucleotides of separation, described polynucleotides are selected from one of the following：
A) nucleotide sequence shown in SEQ ID NO.4 383bp to 2347bp；Or
B) the complementary nucleotide sequence of nucleotide sequence defined in and a).
To realize above-mentioned second purpose, the present invention adopts the technical scheme that：
Polynucleotides as described above are used for the purposes as promoter element.
Described promoter element is used to instruct destination gene expression.
Described target gene comes from algae.
To realize above-mentioned 3rd purpose, the present invention adopts the technical scheme that：
A kind of recombinant vector, described recombinant vector contains polynucleotides as described above, as promoter element.
Described recombinant vector is also containing the target gene being operably connected with the polynucleotides.
Described target gene is located at the downstream of the polynucleotides.
Described recombinant vector is carrier for expression of eukaryon.
To realize above-mentioned 4th purpose, the present invention adopts the technical scheme that：
A kind of genetically engineered host cell, described host cell：
A) containing as above any described recombinant vector；Or
B) polynucleotides as described above of external source are integrated with its genome.
Described host cell is fungi.
The invention has the advantages that：
The experience that studies for a long period of time of the invention based on inventor, the FAD genes of edge green alga Δ 6 are incised according to what is be cloned into
CDNA full length sequences (accession number JN205755) design primer, using genomic walking (genome walking) kit certainly
Incise 5 '-flanking sequence that a length of 2347bp of FAD upstream region of gene of Δ 6 is arrived in amplification in the genomic DNA of edge green alga.Believe in biology
On the basis of ceasing credit analysis, the promoter Analysis and prediction experience enriched with reference to inventor, wherein long 1965bp fragment is connected
Enter to cut off T7 promoters and insert in the pYES2 carriers of green fluorescent protein (GFP) gene, be then transferred to uracil synthesis defect
In the saccharomyces cerevisiae BY4741 of type.After induced expression, transgenic yeast is observed using laser confocal microscope (Confocal)
Result show that the long 1965bp sequences being transferred to can start GFP gene expressions downstream.The knot of Fluorescence Intensity Assays
Fruit shows that it has similar starting efficiency to T7 promoters.The long 1965bp sequences that this result of study shows to be transferred to are scarce
The promoter of the FAD genes of edge green alga Δ 6 is carved, and can with high intensity start the great expression of target gene, is artificial adjustment algae
The isogenic expression of class desaturase creates condition.
Brief description of the drawings
Accompanying drawing 1 is gene walking three-wheel pcr amplification product electrophoretogram.Swimming lane M1 and M2：Respectively D2000 and Maker IV
Molecular weight standard；1st, 2,3 swimming lanes correspond respectively to the FAD 5 '-flanking sequence fragments of upstream region of gene of Δ 6 the 1st, 2,3 wheel PCR
Amplified production.
Accompanying drawing 2 is pMD19-T Vector maps.
Accompanying drawing 3 is the long 1965bp of FAD upstream region of gene of Δ 65 '-flanking sequence and the controlling element predicted.
Accompanying drawing 4 is pCAMBIA1304 binary expression vector collection of illustrative plates.
Accompanying drawing 5 is pYES2 Vector maps.
Accompanying drawing 6 is the laser copolymerization for having converted pY-DT7-1965-GFP, pY-DT7-GFP and pY-GFP brewing yeast cell
Focusing microscope collection of illustrative plates.Wherein, Y-DT7-GFP transgenic yeasts are negative control group, and Y-GFP transgenic yeasts are positive control
Group；GFP is the green fluorescent protein signal graph absorbed under 485nm exciting lights.Scale in figure is 5 μm of sizes.
Accompanying drawing 7 is the comparison of transgenic yeast fluorescence intensity.
Embodiment
Embodiment provided by the invention is elaborated below in conjunction with the accompanying drawings.
Embodiment 1
Primary operational of the present invention includes：
(1) edge green alga will be incised to be inoculated in the triangular pyramidal bottle of BG-11 culture mediums, be placed in temperature for 25 DEG C, illumination it is strong
Spend for 115 μm of ol photons m-2s-1, periodicity of illumination be illumination:Dark/12h:Cultivated in 12h incubator, periodically daily
Shaken several times.Frond cell is collected, and extracts genome DNA.
(2) it is special according to the FAD gene cDNA full length sequences (accession number JN205755) of edge green alga Δ 6 design gene is incised
Different primer (GSP1：SEQ ID NO.1；GSP2：SEQ ID NO.2；GSP3：SEQ ID NO.3), pass through genomic walking technology
5 '-flanking sequence (SEQ ID NO.4) of the FAD upstream region of gene of Δ 6 is cloned, then promoter sequence is carried out by the webserver
The controlling element on-line prediction of row.
(3) according to the acquired 5 '-flanking sequence of FAD upstream region of gene of Δ 6 and the analysis result design of combination promoter sequence
(SEQ ID NO.5 and SEQ ID NO.6, wherein SEQ ID NO.5 1-6bp are band KpnI/BamHI restriction enzyme sites primer
KpnI restriction enzyme sites, SEQ ID NO.6 1-6bp is BamHI restriction enzyme sites), reacted using PCR on the FAD genes of Δ 6
The long 1965bp of 5 '-flanking sequence of trip fragment is subcloned into pMD19-T carriers, obtains recombinant plasmid pMD19T-1965.It is based on
The coded sequence design band BamHI/EcoRI restriction enzyme site primers (SEQ of GFP genes in pCAMBIA1304 binary expression vectors
ID NO.7 and SEQ ID NO.8, wherein SEQ ID NO.7 1-6bp are BamHI restriction enzyme sites, the of SEQ ID NO.8
1-6bp is EcoRI restriction enzyme sites), clone the sequence of GFP genes using pcr amplification reaction and build pMD19T-GFP clone's matter
Grain.
(4) double digestion (BamHI/EcoRI) is carried out to pYES2 carriers and pMD19T-GFP plasmids, reclaims GFP target gene
Fragment, then connected by the use of T4DNA ligases to obtain recombinant expression carrier pY-GFP (as positive control).
(5) double digestion (SpeI/KpnI) excision T7 promoter fragments are carried out to carrier pY-GFP, then with T4DNA ligases
Connection obtains not containing the carrier pY-DT7-GFP of T7 promoters (as negative control).
(6) double digestion (KpnI/BamHI) is carried out to pY-DT7-GFP carriers and pMD19T-1965 plasmids, reclaims purpose piece
Section, then connected with T4DNA ligases to obtain recombinant expression carrier pY-DT7-1965-GFP.
(7) recombinant expression carrier pY-DT7-1965-GFP and pY-DT7-GFP and pY-GFP carrier are turned with electric shocking method
Change yeast defect strain BY4741, screen transformant using the synthetic media (SC-U) of uracil-deficient, obtain Y-DT7-1965-
3 plants of transgenic yeasts such as GFP, Y-DT7-GFP and Y-GFP.
(8) transgenic yeast and wild-type yeast BY4741 are inoculated in SC-U culture mediums, half is carried out to bacterial strain Y-GFP
Lactose Fiber differentiation, other bacterial strains Y-DT7-1965-GFP and Y-DT7-GFP glucose cultures, after 72h under Confocal
Detection.It was found that Y-DT7-GFP transgenic yeast bacterial strains do not have fluorescence, and 2 plants of transgenosis ferment such as Y-GFP and Y-DT7-1965-GFP
Mother has very strong fluorescence.
(9) in the case where being transferred to the cell density of yeast strain unanimously, it is strong to determine respective fluorescence using ELIASA
Degree, analyzed using SPSS statistical softwares, it is found that the fluorescence intensity otherness between bacterial strain Y-DT7-1965-GFP and Y-GFP does not show
Write (P>0.05) the fluorescence intensity otherness extremely significantly (P, but between bacterial strain Y-DT7-GFP<0.01).So as to prove to incise certainly
The FAD genes 5 ' of Δ 6 being cloned into edge green alga-upper brigade commander 1965bp sequence has the function of promoter.
Concrete operations are as follows：
1st, material
(1) incise edge green alga (Myrmecia incisa Reisigl H4301) and be purchased from Prague, CZE Charles university
Algae culture center (CAUP).The algae is inoculated in the triangular pyramidal bottle containing BG-11 culture mediums, be placed in temperature for 25 DEG C,
Intensity of illumination is 115 μm of ol photons m-2s-1, periodicity of illumination be illumination:Dark/12h:Cultivated in 12h incubator, often
Its regular shaken several times.
(2) pYES2 carriers are purchased from Invitrogen companies.Auxotrophy strain yeast BY4741 (is His–、Leu–、Met–And
Ura–Auxotroph, it is easy to the screening of transgenic yeast) it is purchased from Swedish Agricultural University of Science and Technology Dr.Stymne laboratories.By ferment
Mother is inoculated in SC culture mediums, with 200 revs/min of (rpm) speed oscillation cultures at 28 DEG C.
2nd, method
(1) frustule for taking 100mg fresh is put into the mortar of precooling, is added liquid nitrogen and is fully ground.
(2) using the genomic DNA of cetyl trimethylammonium bromide (CTAB) method extraction frustule, -20 DEG C are placed in
Save backup.
(3) based on cDNA full length sequences (the GenBank accession number for incising the FAD genes of edge green alga Δ 6：JN205755), if
Count 3 gene specific primer GSP1 (SEQ ID NO.1), GSP2 (SEQ ID NO.2) and GSP3 (SEQ ID NO.3).Primer
Designing the principle observed is：Three primers are the anti-sense primer of sequence always, with known array reverse complemental, GSP2 position
It should design and be located at GSP2 inner side in GSP1 inner side, GSP3；The distance between each two primer is advisable with 60~100bp；Draw
The length of thing is 22 to 26bp；Tm values are between 60 DEG C to 70 DEG C, G/C content 45% to 55%.
(4) the use of the edge green alga genomic DNA of incising extracted is template, carries out three-wheel PCR.First round PCR system is
Incise the μ L of edge green alga genomic DNA 1, μ L of dNTP Mixture 8, the μ L of 10 × LA PCR Buffer II 5, the μ of LA Taq 0.5
L, μ L of AP Primer 1, μ L of GSP1 primers 1 plus dH2O to 50 μ L.PCR reaction systems are：94 DEG C of pre-degeneration 1min；98 DEG C pre-
It is denatured 1min；5 circulations include 94 DEG C of denaturation 30s, 65 DEG C of annealing 1min, 72 DEG C of extension 2min；94 DEG C of denaturation 30s, 25 DEG C are moved back
Fiery 3min, 72 DEG C of extension 2min；15 circulations include：94 DEG C of denaturation 30s, 65 DEG C of annealing 1min, 72 DEG C of extension 2min；94 DEG C of changes
Property 30s, 65 DEG C annealing 1min, 72 DEG C extension 2min；94 DEG C of denaturation 30s, 44 DEG C of annealing 1min, 72 DEG C of extension 2min；Last 72
DEG C extension 10min.
(5) second wheels and third round PCR are carried out using the first round and the second wheel PCR primer as template respectively, and reaction system is：
The first round or μ L, the LA Taq 0.5 of the second 8 μ L, 10 × LA PCR Buffer II of wheel 1 μ L, dNTP Mixture of PCR primer 5
The μ L of 1 μ L, GSP2 or GSP3 primers of μ L, AP Primer 1, add dH2O to 50 μ L.PCR reaction systems are：15 circulations include 94
DEG C denaturation 30s, 65 DEG C annealing 1min, 72 DEG C extension 2min；94 DEG C of denaturation 30s, 65 DEG C of annealing 1min, 72 DEG C of extension 2min；94
DEG C denaturation 30s, 44 DEG C annealing 1min, 72 DEG C extension 2min；Last 72 DEG C of extensions 10min.
(6) three-wheel PCR primer is detected (Fig. 1) by agarose gel electrophoresis respectively, and third round PCR is obtained
Single band rubber tapping recovery purifying, be then attached to pMD19-T carriers (Fig. 2) and be transformed into Escherichia coli (Escherichia
Coli) DH5 α competent cells, blue hickie screening is carried out, picking positive colony, life work life in Shanghai is sent to after bacterium colony PCR checkings
Thing engineering company is sequenced.The sequence redesigns primer checking after sequencing, so as to ensure the long 2347bp sequences obtained
(SEQ ID NO.4) is to incise the accurate 5 '-flanking sequence of FAD upstream region of gene of edge green alga Δ 6.
(7) the promoter core space of the long 2347bp sequences obtained using network data base prediction and transcription initiation position
Point, using software prediction promoter feature member, respectively positioned at two element the TATA box and CAAT at -24 places and -64 places
Box constitutes the core sequence of promoter, additionally predicted nitrogen controlling element NIT 2, low temperature induction Expression element LTR,
Protein metabolism controlling element O2-site and 5 controlling element GT1-motif, GCN4_motifs related to light reaction, Sp1,
MNF1, CATT-motif etc. (Fig. 3).Use software prediction to tandem repetitive sequence (ACT)6(Fig. 3).These prediction results are shown
Promoter possessed primary element and some specific controlling elements, sequence in addition are contained within 1965bp
Row are almost not previously predicted regulating element.So whether the sequence that we inquire into this segment length 1965bp has promoter function.
(8) the bioinformatic analysis result design band of sequence and promoter sequence based on obtained a length of 2347bp
KpnI/BamHI restriction enzyme sites primer (SEQ ID NO.5 and SEQ ID NO.6) enters performing PCR amplification.PCR response procedures are 94 DEG C
Pre-degeneration 3min, 35 circulations include 94 DEG C of denaturation 45s, 62 DEG C of annealing 45s, 72 DEG C of extension 2min, last 72 DEG C of extensions
10min.PCR primer is verified and is sequenced through glue reclaim, clone, the screening of blue hickie, bacterium colony PCR as stated above, obtains incising edge
The FAD genes 5 ' of green alga Δ 6-upper brigade commander 1965bp sequence.PMD19T- is extracted from being sequenced in accurate bacillus coli DH 5 alpha
1965 plasmids, -20 DEG C save backup.
(9) clone of GFP gene orders.Designed according to the coded sequence of GFP genes on pCAMBIA1304 carriers (Fig. 4)
Band BamHI/EcoRI restriction enzyme sites primer (SEQ ID NO.7 and SEQ ID NO.8) enters performing PCR amplification.PCR response procedures are：
94 DEG C of pre-degeneration 3min, 35 circulations include 94 DEG C of denaturation 45s, 57 DEG C of annealing 45s, 72 DEG C of extension 1min, last 72 DEG C of extensions
10min.PCR primer is verified and is sequenced through glue reclaim, clone, the screening of blue hickie, bacterium colony PCR as stated above, obtains encoding GFP
The fragment of gene.PMD19T-GFP plasmids are extracted from being sequenced in accurate bacillus coli DH 5 alpha, 20 DEG C save backup.
(10) BamHI and EcoRI double digestion pYES2 expression vectors (Fig. 5) and recombinant plasmid pMD19T-GFP are used.Reactant
It is to be：Each μ L of 1 μ L, 10 × K buffer solution 2 of 2 kinds of enzymes, 6 μ L carrier DNA, add water to 20 μ L.37 DEG C of digestion 4h.Utilize 1% agar
GFP genetic fragments and pYES2 carrier segments after sugared electrophoresis detection digestion products and glue reclaim digestion, make in T4DNA ligases
With lower 16 DEG C connections overnight, construction of expression vector pY-GFP (as positive control).It is thin to be transformed into bacillus coli DH 5 alpha competence
Born of the same parents, transformant is screened with the LB resistance cultures base of the Amp containing 50mg/L.Next day selects several single bacterium colonies, shaking table culture, uses alkali cracking
Solution extracts DNA, and plasmid is identified by BamHI and EcoRI double digestions and pcr amplification reaction, and detection is correctly positive
Clone serves the raw work sequencing in sea.Sequence determine it is errorless after, cultivate and preserve strain.
(11) SpeI and KpnI double digestion pY-GFP expression vectors are used.Reaction system is：2 kinds of enzymes each 1 μ L, 10 × M buffering
The μ L of liquid 2,6 μ L carrier DNA, add water to 20 μ L.37 DEG C of digestion 4h.Returned using 1% agarose electrophoresis detection digestion products and glue
Purpose fragment is received, overnight, structure does not contain the expression vector of T7 promoter sequences for 16 DEG C of connections under T4DNA connection enzyme effects
PY-DT7-GFP (as negative control).Positive colony is screened using above method, then will detect correct positive colony
Serve the raw work sequencing in sea.Sequence determine it is errorless after, cultivate and preserve strain.
(12) KpnI and BamHI double digestion pY-DT7-GFP expression vectors and recombinant plasmid pMD19T-1965 are used.Reactant
It is to be：Each μ L of 1 μ L, 10 × K buffer solution 1 of 2 kinds of enzymes, 6 μ L carrier DNA, add water to 20 μ L.37 DEG C of digestion 4h.Utilize 1% agar
The purpose fragment and pY-DT7-GFP carrier segments that 1965bp grows after sugared electrophoresis detection digestion products and glue reclaim digestion,
The lower 16 DEG C of connections of T4DNA connections enzyme effect are stayed overnight, construction of expression vector pY-DT7-1965-GFP.Screened using above method
Positive colony, it then will detect correct positive colony and serve the raw work sequencing in sea.Sequence determine it is errorless after, cultivate and preserve bacterium
Kind.PY-GFP, pY-DT7-GFP and pY-DT7-1965-GFP plasmid are extracted from bacillus coli DH 5 alpha, 20 DEG C save backup.
(13) preparation of competent yeast cells.By 1:1000 are inoculated in yeast BY4741 in SC culture mediums, and 28 DEG C multiple
Soviet Union's overnight incubation, then by 1:100 amplification cultures, are about 1 × 10 with 200rpm speed oscillations culture to cell density8cells/
ML (about 4~5h).Cooled on ice 15min makes cell stop growing, and 5min is centrifuged with 5000rpm rotating speeds under the conditions of 4 DEG C, collects ferment
Mother cell, the sterile water washing cell of precooling 3 times, it is collected by centrifugation under similarity condition.The 1M sorbitol washes cell 1 of 20mL precoolings
It is secondary, the 1M sorbierites of 0.5mL precoolings are then dissolved in, adjust the density of cell about 1 × 1010cells/mL.Cell is preserved on ice,
It is easy to electric shock to use.
(14) electric shocking method transformed yeast cell BY4741 is utilized.Take in precooling on ice pY-GFP, pY-DT7- to be transformed
The μ L of GFP and pY-DT7-1965-GFP DNAs about 5~10 (content about 5~200ng) mix with competent cell, and with
The precooling on ice together of 0.2cm electric shock cup, then DNA and cell mixed liquor are transferred in the electric shock cup of precooling and gently mixed
Even, after ice bath 5min, in electroporation apparatus (Bio-Rad), option program Sc2 shocks by electricity once, by these recombinant expression carriers point
It is not transformed into yeast BY4741 competent cells.Electric shock cup is removed after electric shock, is added immediately the 1M sorbierites of 1mL precoolings, gently
Gently it is transferred in new YPD culture mediums, after 28 DEG C of slight oscillatory 5h, bacterium solution is coated on the uracil-deficient containing 1M sorbierites
On synthetic media (i.e. SC-U), 28 DEG C of inversion quiescent culture 48-72h, picking colony cultivates 48- in liquid SC-U culture mediums
72h.Y-GFP, Y-DT7-1965-GFP and Y-DT7-GFP transgenic yeast are after colony PCR amplification reaction checking, with containing 20%
The SC-U culture mediums (containing 2% glucose) of glycerine preserve.
(15) laser confocal fluorescence microscope observation yeast cells is utilized.By the transgenic yeast of above-mentioned preservation through 72h
After induced expression, draw appropriate bacterium solution and make slide, existed using laser confocal fluorescence microscope (Carl Zeiss companies)
The green florescent signal of GFP in yeast cells is detected under 485nm exciting lights, its cellular morphology is observed under light field and by itself and fluorescence
Signal graph integrates (Fig. 6).
(16) fluorescence intensity of ELIASA detection yeast cells is utilized.Use ultramicrospectrophotometer (Thermo
Scientific, USA) by the optical density (OD of living cells bacterium solution after above-mentioned induced expression culture600) value is tuned into unanimously, respectively plus
Three kinds of appropriate bacterium solutions are to (DragonLab) on the orifice plate of black 96, using ELIASA (BioTek, USA) fluorescence intensity, often
Individual sample is repeated 3 times, and is used statistic software SPSS analyze data (Fig. 7).
3rd, result
(1) genomic walking technology is utilized, clone obtains incising edge green alga Δ 6 by three-wheel pcr amplification reaction (Fig. 1)
5 '-flanking sequence of FAD upstream region of gene, and react and be sequenced by PCR through redesigning primer, it is 2347bp to confirm its length
(SEQ ID NO.4)。
(2) there is this characteristic of multiple cloning sites using T7 promoters downstream on pYES2 expression vectors, pass through restriction enzyme
The endonuclease reaction of enzyme, the downstream of obtained GFP genetic fragments insertion T7 promoters will be cloned.T7 promoters, and root are cut off afterwards
According to bioinformatics to the analysis result (Fig. 3) for incising the FAD 5 '-flanking sequences of upstream region of gene of edge green alga Δ 6 that is cloned into,
The sequence of wherein long 1965bp (at SEQ ID NO.4 383bp to 2347bp) is connected to GFP upstreams, with substitution
T7 promoters on pYES2 expression vectors, thus construct to include and incise the FAD gene promoters of edge green alga Δ 6 and GFP
Expression plasmid.The plasmid built is transferred in saccharomyces cerevisiae by electroporated method, laser confocal fluorescence microscope
Testing result (Fig. 6) is shown, the 5 '-flanking sequence for incising the long 1965bp of FAD upstream region of gene of edge green alga Δ 6 is carried being transferred to
In plasmid pY-DT7-1965-GFP yeast cells, and in the yeast for being transferred to carrier pY-GFP of the carrying containing T7 promoters
In cell, GFP green florescent signal can be detected；And in the yeast for the carrier pY-DT7-GFP for being transferred to excision T7 promoters
GFP green florescent signal is not detected in cell.Show to clone to obtain incises the FAD upstream region of gene of edge green alga Δ 6 length
The 5 ' of 1965bp-flanking sequence has the function that can start downstream GFP gene expressions.The statistics of transgenic yeast fluorescence intensity
Analysis result (Fig. 7) shows that the fluorescence intensity between Y-DT7-1965-GFP and Y-GFP this 2 plants of transgenic yeast cells does not have
Substantially (P>0.05) difference, their two pole significantly (P<0.01) yeast for being different from conversion pY-DT7-GFP plasmids is thin
Born of the same parents.So as to show that the 5 '-flanking sequence for incising the long 1965bp of edge green alga Δ 6FAD upstream region of gene opens with T7 on pYES2 expression vectors
Mover has the same function of efficiently starting downstream gene expression.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of polynucleotides of separation, it is characterised in that described polynucleotides are selected from one of the following：
A) nucleotide sequence shown in SEQ ID NO.4 383bp to 2347bp；Or
B) the complementary nucleotide sequence of nucleotide sequence defined in and a).
2. polynucleotides as claimed in claim 1 are used for the purposes as promoter element.
3. purposes according to claim 2, it is characterised in that described promoter element is used to instruct target gene table
Reach.
4. purposes according to claim 2, it is characterised in that target gene comes from algae.
5. a kind of recombinant vector, it is characterised in that described recombinant vector contains polynucleotides as claimed in claim 1, makees
For promoter element.
6. recombinant vector according to claim 5, it is characterised in that described recombinant vector also contains and more nucleosides
The target gene that acid is operably connected.
7. recombinant vector according to claim 5, it is characterised in that target gene is located at the downstream of the polynucleotides.
8. recombinant vector according to claim 5, it is characterised in that described recombinant vector is carrier for expression of eukaryon.
A kind of 9. genetically engineered host cell, it is characterised in that described host cell：
A) containing the recombinant vector as described in claim 5-8 is any；Or
B) polynucleotides as claimed in claim 1 of external source are integrated with its genome.
10. host cell according to claim 9, it is characterised in that described host cell is fungi.
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| CN1810975A (en) * | 2006-02-21 | 2006-08-02 | 南开大学 | Delta 6-fatty acid dehydrogenase promoter sequence of Thamnidium elegans and its application |
| CN101955954A (en) * | 2010-09-10 | 2011-01-26 | 兰州大学 | Microula sikkimensis delta-6 fatty acid desaturase gene (Ms-delta6-FAD) and application thereof |
| CN102943081A (en) * | 2012-11-22 | 2013-02-27 | 上海海洋大学 | Deoxyribose nucleic acid (DNA) sequence for encoding parietchloris incise diacylglycerol acyltransferase and application thereof |
| CN103756985A (en) * | 2013-12-11 | 2014-04-30 | 上海海洋大学 | Myrmecia incisa Reisigl diacylglycerol acyltransferase gene sequence and use thereof |
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2015
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1810975A (en) * | 2006-02-21 | 2006-08-02 | 南开大学 | Delta 6-fatty acid dehydrogenase promoter sequence of Thamnidium elegans and its application |
| CN101955954A (en) * | 2010-09-10 | 2011-01-26 | 兰州大学 | Microula sikkimensis delta-6 fatty acid desaturase gene (Ms-delta6-FAD) and application thereof |
| CN102943081A (en) * | 2012-11-22 | 2013-02-27 | 上海海洋大学 | Deoxyribose nucleic acid (DNA) sequence for encoding parietchloris incise diacylglycerol acyltransferase and application thereof |
| CN103756985A (en) * | 2013-12-11 | 2014-04-30 | 上海海洋大学 | Myrmecia incisa Reisigl diacylglycerol acyltransferase gene sequence and use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 缺刻缘绿藻FAD 基因的序列特征及其相对转录量对氮饥饿的响应;刘凡等;《中国水产科学》;20120930;第19卷(第5期);729,730,732,735 * |
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