CN105274068B - It is a kind of to encode DNA sequence dna and its application for incising 9 fatty acid desaturase of edge green alga Δ - Google Patents
It is a kind of to encode DNA sequence dna and its application for incising 9 fatty acid desaturase of edge green alga Δ Download PDFInfo
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Abstract
The present invention relates to DNA sequence dna and its applications that a kind of coding incises 9 fatty acid desaturase of edge green alga Δ.Pass through the screening to incising edge green alga transcript profile high throughput database, obtain the contig sequences of 1 and known species Δ 9FAD DNA homologs, and thus design primer, the full length sequence for incising edge green alga Δ 9FAD genes is obtained using the ends cDNA rapid amplifying technology clone, the heterogenous expression gene removes the ORF of chloroplast transit peptides in the saccharomyces cerevisiae that oleic acid synthesizes deficiency ole1 mutant strains, GC MS analyses are carried out to the aliphatic acid of transgenic yeast, newly generated substance is detected in the transgenosis mutant strain yeast --- palmitoleic acid and oleic acid, to prove that the albumen of the coded by said gene has the function of Δ 9FAD.The present invention is that genetic manipulation is carried out in plant, to realize that PUFA's efficiently synthesizes the condition of creating.
Description
Technical field
The present invention relates to molecular biology and gene engineering technology field, specifically, being related to a kind of coding, to incise edge green
The DNA sequence dna of 9 fatty acid desaturase of algae Δ and its application.
Background technology
There is a large amount of polyunsaturated fatty acid (polyunsaturated fatty acid, PUFA) in organism,
These PUFA are for maintaining the normal physiological function of organism to play an important roll, according to since the double bond of first, methyl end
PUFA points can be ω -3 systems and ω -6 systems by position.In ω -6 is PUFA, arachidonic acid (arachidonic acid,
C20:4Δ5,8,11,14, AA) and it is one of most representative aliphatic acid.AA category essential fatty acids are in body with important
The precursor of 20 carbon polyenoid derivatives of physiological action, such as prostaglandin E2, prostacyclin and leukotriene.AA or one kind are important
Nutraceutical, in terms of brain and visual acuity have special role, therefore be added to extensively infant food formula in.
In addition, AA or a kind of cosmetics of super quality raw material, are added in some cosmetic skincare products and hair-dressing article, can play
The effect of keeping skin wet, delay skin aging and prevention and treatment alopecia.
The traditional source of AA is deep sea fish oil and animal viscera.But AA contents are very low in animal tissue, about 0.2% He
0.5%, the AA prepared in this way is expensive, it is difficult to meet the needs in market.Abroad in Recent Years starts
Raw material produces AA, for example, a commerciality bacterial strain 1S-4 of Mortierella alpina (Mortieralla alpina) has been used for work
Industry fermenting and producing AA.And the discovery of edge green alga (Myrmecia incisa Reisigl) is incised, to utilize bioreactor
Large-scale industrial production AA provides new thinking.
It is monoplast green alga to incise edge green alga, belongs to Chlorophyta, Trebouxiophyceae guiding principles, and cell often flocks together
Form the cell mass of similar non-coenobium.Research finds that the algae contains a large amount of AA, especially under the conditions of nitrogen hunger, content
The 60% of frond total fatty acid content can be reached.
In incising the microalgaes such as edge green alga, the synthesis of AA is often since saturated fatty acid, i.e., from stearic acid to oleic acid
Conversion, which be catalyzed by Δ 9FAD, and first undersaturated double bond is introduced between stearic C9 and C10, generates oil
Acid.Later, oleic acid is along ω -6 approach, then in the work of 6 fatty acid elongase of a series of FAD such as Δ 12, Δ 6 and Δ 5 and Δ
Under, AA is finally synthesized.It can be seen that it is the key that AA synthesizes a step that stearic acid desaturation, which is generated oleic acid, by Δ 9FAD.
Clone and identification, which obtain, incises edge green alga Δ 9FAD encoding genes for carrying out genetic manipulation in plant, with reality
Existing efficiently synthesizing for PUFA is of great significance.
Invention content
The purpose of the present invention is being directed to deficiency in the prior art, a kind of polypeptide is provided.
Another purpose of the present invention is to provide a kind of nucleotide sequence of separation.
Another purpose of the present invention is to provide a kind of recombinant expression carrier.
Fourth object of the present invention is to provide a kind of genetically engineered host cell.
The 5th purpose of the present invention is to provide polypeptide as described above, nucleotide sequence as described above, as described above
Recombinant expression carrier or host cell as described above purposes.
To realize above-mentioned first purpose, the technical solution adopted by the present invention is that：
A kind of polypeptide, 62-432 or SEQ ID of the amino acid sequence such as SEQ ID NO.15 of the polypeptide
Shown in NO.15.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that：
A kind of nucleotide sequence of separation, the nucleotide sequence include：
A) 158bp to 1453bp, SEQ of the 341bp to 1453bp, SEQ ID NO.3 of SEQ ID NO.3
Nucleotide sequence shown in ID NO.3 or SEQ ID NO.12；Or
B) nucleotide sequence of nucleotide sequence complementation shown in and a).
To realize above-mentioned third purpose, the technical solution adopted by the present invention is that：
A kind of recombinant expression carrier, the recombinant expression carrier are by nucleotide sequence as described above and plasmid or disease
Recombinant expression carrier constructed by poison.
As a kind of specific implementation mode of the present invention, the plasmid is pYES2 plasmids.
To realize above-mentioned 4th purpose, the technical solution adopted by the present invention is that：
A kind of genetically engineered host cell, the host cell are selected from one of the following：
A) host cell for being converted or being transduceed with nucleotide sequence as described above；
B) host cell for being converted or being transduceed with recombinant expression carrier as described above.
As a kind of specific implementation mode of the present invention, the host cell is that bacterial cell, fungal cell, plant are thin
Born of the same parents or zooblast.
As a kind of specific implementation mode of the present invention, the host cell is yeast cells.
To realize above-mentioned 5th purpose, the technical solution adopted by the present invention is that：
Polypeptide, nucleotide sequence as described above, recombinant expression carrier as described above or as described above as described above
Purposes of the host cell in synthesizing polyunsaturated fatty acid.
Polypeptide, nucleotide sequence as described above, recombinant expression carrier as described above or as described above as described above
Purposes of the host cell in being palmitoleic acid by palmitic acid desaturation or being oleic acid by stearic acid desaturation.
The invention has the advantages that：
The present invention obtains 1 and known species Δ by the screening to incising edge green alga transcript profile high throughput database
The contig sequences (Contig16329) of 9FAD DNA homologs, and thus design primer, utilize the ends cDNA rapid amplifying technology
(RACE) clone obtains the full length sequence for incising edge green alga Δ 9FAD genes.The wine brewing of deficiency ole1 mutant strains is synthesized in oleic acid
The heterogenous expression gene removes the ORF of chloroplast transit peptides in yeast, and GC-MS analyses are carried out to the aliphatic acid of transgenic yeast,
Newly generated substance is detected in the transgenosis mutant strain yeast --- palmitoleic acid and oleic acid, to prove the coded by said gene
Albumen have the function of Δ 9FAD, and desaturation is efficient.The present invention is that genetic manipulation is carried out in plant, to realize
PUFA's efficiently synthesizes the condition of creating.
Description of the drawings
Fig. 1 Δ 9FAD gene cDNAs and DNA sequence dna amplified production electrophoretogram (A) and its gene structure (B).Swimming lane M：DL
2000 molecular weight standards；Swimming lane 1：5 '-RACE end products；Swimming lane 2：3 '-RACE end products；Swimming lane 3 to 6：It is profit respectively
Gene DNA sequence is carried out with primer pair Seq 4 and Seq 5, Seq 6 and Seq7, Seq 8 and Seq 9 and Seq 10 and Seq 11
Amplified production.Gray line and black line are respectively UTR and introne, and black surround is exon.
Fig. 2 .pMD19-T Vector maps.
Fig. 3 .pYES2 Vector maps.
The gas-chromatography and GC-MS collection of illustrative plates of Fig. 4 yeast fat sour components.A：Gas chromatogram；B、C：Respectively scheme A-c
In occur the MS collection of illustrative plates at two novel substance peaks from left to right.a：Ole1 saltant comparison groups；b：Turn empty vector control group；c：Turn to lack
Carve the yeast Y- Δs 9FAD of edge green alga Δ 9FAD genes.
Specific implementation mode
It elaborates below in conjunction with the accompanying drawings to specific implementation mode provided by the invention.
Embodiment 1
Primary operational of the present invention includes：
1) edge green alga is incised temperature is 25 DEG C, intensity of illumination is 115 μm of ol m-2s-1Under conditions of, it is cultivated in BG-11
It is cultivated in base, the photoperiod is illumination 14h:Dark 10h, aperiodically rocks daily.Frustule is collected, RNA and DNA is extracted.
2) by the screening to incising edge green alga transcript profile high throughput database, 1 contig sequence is obtained
(Contig16329), according to this sequence design gene specific primer Seq 1 and Seq 2, Clontech companies RACE skills are utilized
Art carries out 5 '-ends and the amplification of 3 '-end fragments respectively.It recycles this two segment and is cloned on pMD19-T, then convert to large intestine
Bacillus is screened with after sequencing through blue hickie, with former contig sequence assemblies and carries out PCR by redesign primer and react and survey
Sequence is verified, and the full length sequence (Seq 3) of Δ 9FAD gene cDNAs is obtained.
3) bioinformatic analysis is carried out to the Δ 9FAD gene cDNAs full length sequence (Seq 3) of acquisition, finds its opening
Reading frame (ORF) is 1299bp, and four couples of gene specific primer Seq 4 and Seq 5,6 and of Seq are designed according to cDNA full length sequences
Seq 7, Seq 8 and Seq 9 and Seq 10 and Seq 11 expand introne.PCR product is recycled, is sequenced as stated above
Verify and with former sequence assembly, obtain the DNA sequence dna (Seq 12) of Δ 9FAD genes.
4) by bioinformatic analysis it is found that there are 1 in its aminoterminal for protein sequence coded by Δ 9FAD genes ORF
The chloroplast transit peptides being made of 61 amino acid.The ORF of chloroplast transit peptides is free of according to pYES2 carriers and Δ 9FAD genes
Primers Seq 13 and Seq 14 (restriction enzyme site containing EcoRI/XbaI) carries out PCR amplification, and TA is cloned and through EcoRI/
After XbaI double digestions, T is utilized4Ligase connects to obtain recombinant vector pY- Δs 9FAD.
5) recombinant vector pY- Δs 9FAD is synthesized into deficiency in electroporation apparatus with electric shocking method transformed saccharomyces cerevisiae oleic acid
Ole1 mutant strain BY4389 (His–、Leu–And Ura–Deficiency screens convenient for transgenic yeast), with containing 0.005% linoleic acid
(LA) uracil-deficient synthetic media (SC-U) screening obtains transgenic yeast Y- Δs 9FAD.
6) transgenosis and the yeast-inoculated for turning empty plasmid are added into appropriate LA, in 2% glucose YPD in YPD culture mediums
28 DEG C of culture 3d, go in the YPD culture mediums without LA after reaching certain biomass, add 2% galactolipin 25 in culture medium
The slow-speed of revolution induces 3d under the conditions of DEG C.Collect yeast, freeze-drying.Aliphatic acid directly is carried out using methanol-sulfuric acid method to yeast
Esterification.
7) gas chromatography mass spectrometry (GC-MS) is utilized to detect palmitic acid in the yeast cells for turning to incise edge green alga Δ 9FAD genes
With stearic desaturation product --- it is respectively palmitoleic acid and oleic acid, illustrates that the yeast BY4389 of ole1 mutation is being transferred to this
After gene, restore the synthesis capability of palmitoleic acid and oleic acid.To prove that the coded by said gene albumen has the function of Δ 9FAD.
Concrete operations are as follows：
1, material
1) it incises edge green alga (Myrmecia incisa Reisigl) H4301 and is purchased from Culture collection of
algae of Charles University of Prague(CAUP).Temperature is 25 DEG C, intensity of illumination is 115 μm of ol
photons m-2s-1, light dark ratio be 14h/10h under conditions of cultivate, culture medium BG-11.
2) this laboratory pYES2 preserves, and oleic acid synthesizes deficiency ole1 mutant strain yeast BY4389 (His–、Leu–And Ura–
Deficiency) it is purchased from Osaka, Japan university.Yeast is in YPD culture mediums, with 200 turns/min (rpm) speed oscillation culture at 28 DEG C.
2, method
1) frustule for taking 100mg fresh is placed in the mortar of precooling, and liquid nitrogen is added and is fully ground.
2) TRIzol (Invitrogen companies) method extracted total RNA, -20 DEG C save backup.Take 1-5 μ g RNA reverse transcriptions
Kit (TaKaRa companies) carries out the synthesis of the first chains of cDNA, the PCR reaction templates as gene cloning.Utilize Plant Genome
DNA extraction kit (Tiangeng company) extracts DNA, the PCR reaction templates as amplification introne.
3) basis incises in edge green alga transcript profile high throughput library and screens 1 and known species Δ 9FAD DNA homologs certainly
Contig sequences (Contig16329) design gene-specific primer (gene specific primers, GSP) Seq 1
(SEQ ID NO.1) and Seq 2 (SEQ ID NO.2), according to SMARTTM(Clontech is public for RACE cDNA amplification kits
Department) operation instruction establish 5 '-RACE and 3 '-RACE reverse transcription systems respectively, then expanded using touchdown PCR principle.
5 '-RACE the reaction systems of 25 μ L include：The PCR grades water of 17 μ L, 10 × Advantage 2PCR buffer solutions of 2.5 μ L, 0.5 μ L
DNTP, 0.5 μ L primers (Seq 1), 10 × UPM of 2.5 μ L, 5 '-RACE cDNA of 1.5 μ L, 0.5 μ L 50 ×
2 polymerase mix of Advantage.The reaction system at 3 '-ends, except 3 '-RACE of the primer (Seq2) and 1.5 μ L of 0.5 μ L
It is other with 5 '-end reaction systems outside cDNA.Response procedures：94 DEG C of denaturation 30s, 72 DEG C of extension 150s, 5 recycle；94 DEG C of changes
Property 30s, 70 DEG C annealing 30s, 72 DEG C extension 150s, 5 cycle；94 DEG C of denaturation 30s, 68 DEG C of annealing 30s, 72 DEG C of extension 150s,
30 cycles；72 DEG C of extension 7min.Then, electrophoresis (Fig. 1) is carried out to RACE products and glue recycles, be then attached to pMD19-T
Carrier (Fig. 2) is simultaneously transformed into Escherichia coli (Escherichia coli) DH5 α competent cells, carries out blue hickie screening, picking
Positive colony is sent to the sequencing of Shanghai Sheng Gong bio-engineering corporations after bacterium colony PCR verifications.5 '-and the 3 '-RACE that sequencing is obtained
The sequence of segment and known segment is spliced, and design primer and sequence verification again, has obtained the overall length of Δ 9FAD genes
CDNA sequence (Seq3, SEQ ID NO.3).
Sequence 2442bp, wherein 5 '-non-translational regions (UTR) and 3 '-UTR are respectively 157bp and 986bp, open reading
Reading frame (ORF) a length of 1299bp encodes 432 amino acid residues (Seq 15, SEQ ID NO.15).According to full-length cDNA sequence
Row four pairs of gene specific primer Seq 4 (SEQ ID NO.4) of design and Seq 5 (SEQ ID NO.5), (the SEQ ID of Seq 6
) and Seq 7 (SEQ ID NO.7), Seq 8 (SEQ ID NO.8) and Seq 9 (SEQ ID NO.9) and 10 (SEQ of Seq NO.6
ID NO.10) and Seq 11 (SEQ ID NO.11), introne amplification is carried out respectively.Reaction system includes：1.0 μ L DNA moulds
Plate, primer each 0.5 μ L, 10.5 μ L without RNase water, 12.5 μ L 2 × Taq PCR Master Mix.PCR reaction conditions：95
DEG C pre-degeneration 5min, 36 cycles include 1min and 72 DEG C of 94 DEG C of denaturation 1min, 68.5 DEG C of annealing extension 3min；Last 72 DEG C
Extend 10min.PCR product will be surveyed as stated above through glue recycling, clone, blue hickie screening, bacterium colony PCR verifications and sequencing
Sequence result is spliced with the original areas ORF, obtains the DNA sequence dna (Seq 12, SEQ ID NO.12) of Δ 9FAD genes.
It is shown using Blastx search results and incises the edge green alga Δ encoded protein in the areas 9FAD gene ORF and Rhein clothing
Algae (Chlamydomonas reinhardtii) stearyl-ACP desaturase albumen has 65% similarity.ChloroP
1.1 Server predict that its N-terminal has the chloroplast transit peptides of 61 amino acid residues composition.In vivo, transit peptides are handles
The target protein synthesized in cytoplasm is transported to the targeting proteins of corresponding organelle, and target protein is when entering organelle, the transhipment
Peptide will be cut off by peptase.Therefore when carrying out the Function Identification of target gene, transit peptide sequence should be excluded.
4) according to the ORF sequences of pYES2 carriers and Δ 9FAD genes without chloroplast transit peptides, (341bp of Seq 3 is extremely
1st 453bp) (SEQ ID NO.13,1bp to 2bp are protection base to design primer Seq 13, and 3bp to 8bp is EcoRI
Restriction enzyme site) and Seq 14 (SEQ ID NO.14,1bp to 2bp are protection base, and 3bp to 8bp is XbaI enzyme cutting position
Point).CDNA to incise edge green alga carries out PCR amplification as template.Reaction system is as follows：1.0 μ L cDNA templates, primer Seq
13 and Seq 14 each 0.5 μ L, 10.5 μ L without RNase water, 12.5 μ L 2 × Taq PCR Master Mix.PCR reaction conditions：
95 DEG C of pre-degeneration 5min, 36 cycles are annealed 1min and 72 DEG C comprising 94 DEG C of denaturation 1min, 68.5 DEG C extends 90s, and last 72
DEG C extend 10min.PCR product is as stated above through glue recycling, clone, blue hickie screening, bacterium colony PCR verifications and sequencing, with true
Protect the accuracy of objective gene sequence.Thus obtain pMD19T- Δ 9FAD plasmids.
5) Escherichia coli for carrying correct objective gene sequence are cultivated, then extract pMD19T- Δ 9FAD plasmids,
Double digested reaction is carried out with restriction enzyme EcoRI and XbaI.EcoRI is also carried out to the pYES2 plasmids of extraction simultaneously
It is reacted with XbaI double digestions.Reaction system is 10 × M buffer solutions of 2 μ L, the 0.1%BSA of 4 μ L, DNA 2 μ g, EcoRI and XbaI
Each 1 μ L add no RNase water to 20 μ L.37 DEG C of digestion reaction 4h.It is tapped and recovered purpose piece after product after digestion is carried out electrophoresis
Section, is used in combination T4Δ 9FAD genetic fragments after digestion connect to obtain recombinant vector pY- Δs with pYES2 segments by DNA ligase
9FAD.Coupled reaction system is：The connection buffer solution of 2.5 μ L, Δ 9FAD gene DNA about 0.3pmol, the DNA of carrier pYES2 is about
0.03pmol, the T of 1 μ L4DNA ligase adds no RNase water to 25 μ L, and 16 DEG C of connections are overnight.It is ligated and transformed into Escherichia coli
DH5 α competent cells, using the LB Screening of Media of the benzyl of ammonia containing 50mg/L (Amp), bacterium colony PCR and sequencing are to ensure sequence
Accuracy.PY- Δ 9FAD plasmids are extracted from bacillus coli DH 5 alpha, -20 DEG C save backup.
6) prepared by competent yeast cells.By yeast-inoculated in the YPD culture mediums containing 0.005%LA, 28 DEG C of recovery trainings
It supports overnight, then presses 1:100 amplification cultures, are about 1 × 10 with 250rpm speed oscillations culture to cell density8Cell/mL is (about
4~5h).Cooled on ice 15min makes cell stop growing, thin with 5000rpm rotating speeds centrifugation 5min collection yeast under the conditions of 4 DEG C
Born of the same parents, are pre-chilled sterile water washing cell 2 times, are collected by centrifugation under similarity condition.The 1M sorbitol washes cell of 20mL precoolings 1 time, so
It is dissolved in the 1M sorbierites of 0.5mL precoolings afterwards, adjusts the density of cell 1 × 1010Cell/mL.Cell (or 4 is preserved on ice
DEG C), it is used convenient for electric shock.
7) utilize electroporation apparatus (Bio-Rad) by the electroporated yeast BY4389 competence of carrier pY- Δs 9FAD of recombination
Cell.Take about 5~10 μ L (5~200ng) of pY- Δ 9FAD recombinant plasmid dnas and competence to be transformed being pre-chilled on ice thin
Born of the same parents' mixing, and be pre-chilled on ice together with the electric shock cup of 0.2cm, then electric shock cup that DNA- cell mixtures are transferred to precooling is light
Light mixing, after ice bath 5min, option program Sc2 electric shocks are primary.Electric shock cup is removed, is added immediately the 1M sorbierites of 1mL precoolings, gently
It is gently transferred in new YPD culture mediums, 28 DEG C of slight oscillatory 5h.Bacterium solution is coated on containing 0.005%LA and 1M sorbierites
On uracil-deficient synthetic media (SC-U), 28 DEG C of inversion stationary culture 48-72h, picking colony is in the liquid containing 0.005%LA
It is cultivated in body SC-U culture mediums.After the sequence of target fragment is inserted into bacterium colony PCR verification, with containing 2% glucose or 2% raffinose
SC-U culture mediums preserve strain.
10) bacterium solution of preservation is inoculated in YPD culture mediums, adds appropriate LA, with 220rpm speed oscillation cultures at 28 DEG C.
It is then centrifuged for collecting simultaneously washing thalline, is transferred in the 2% galactolipin YPD culture mediums without LA, with 150rpm rotating speeds at 25 DEG C
Shaken cultivation 72h.Yeast thalline is collected by centrifugation, the liquid nitrogen cryopreservation after deionized water is washed 3 times.
11) yeast of liquid nitrogen cryopreservation is freeze-dried, then carries out methyl esterification of fatty acid.Weigh about 25mg yeast powder in
It is esterified in bottle, the methanol solution that 1mL contains 4% sulfuric acid is added.After inflated with nitrogen, in 85 DEG C of water-bath esterification 1h, convenient for that will tie
The fatty acid molecule of conjunction is fully dissociated and is methylated.After esterification, be separately added into above-mentioned esterification bottle 1mL deionized waters and
1mL n-hexanes, mix well, and then centrifuge 10min at 5500rpm.It collects in supernatant to sample bottle, nitrogen concentration, 4 DEG C
It saves backup.
12) Angilent 7890B type gas chromatographs are used to carry out fatty acid compositional analysis, chromatographic column is HP-88 type hairs
Capillary column (30m × 0.25mm)；Temperature program is 70 DEG C of reservation 1min, and then 10 DEG C/min is warming up to 230 DEG C, finally retains
9min；Do not shunt, nitrogen, hydrogen, air flow velocity be respectively 25mL/min, 35mL/min, 400mL/min.Sample size is 0.2
μL。
13) GC-MS detects newly-generated product.Fatty acid standards and defective yeast are compareed in the yeast of transgenosis
When confirmation has new product to generate, detected using GC-MS.The chromatographic column of GC is HP-INNOWax Polyethylene Glyco
(0.25 μm of the μ ms of 30m × 320) connects MS level four bars, 150C (maximum value 200C).50 DEG C of holding 1min of temperature program, with 10
DEG C/min be raised to 150 DEG C holding 1min, then again with 4 DEG C/min be raised to 230 DEG C keep 5min, run time 37min.Helium is to carry
Gas, 50 DEG C, pressure 7.6522psi, flow velocity 2.6891mL/min, Mean Speed 59.763cm/s of initial value, residence time
0.83664min, flow velocity 2.6891mL/min, run time 37min.MS acquisition parameters select EMV patterns, tracing detection from
Son can be 69.922eV.
3, result
1) it expands to obtain cDNA full length sequences (Seq3, the SEQ ID for incising edge green alga Δ 9FAD genes using RACE methods
NO.3).2 442bp of the sequence, wherein 5 '-UTR and 3 '-UTR are respectively 157bp and 986bp, a length of 1299bp of ORF,
Encode 432 amino acid residues (Seq 15, SEQ ID NO.15).Using DNA as template, amplifies and incised edge green alga Δ 9FAD
The DNA sequence dna (Seq 12, SEQ ID NO.12) of gene.4 198bp of the sequence, there are 8 intrones, they are from 5 '-
End start to be sequentially located at 152-578,852-1025 of the gene DNA sequence, 1182-1460,1625-1850,1939-2081,
At 2178-2350,2503-2659 and 2823-3029, to which the cDNA sequence of the gene is divided into 8 exons, and by 5 '-
UTR is divided into long respectively two sections (B in Fig. 1) of 151bp and 6bp.
2) it selects pYES2 carriers successfully to be constructed by restriction enzyme EcoRI and XbaI and incises edge green alga Δ
The expression vector pY- Δs 9FAD of 9FAD genes.It, can because Δ 9FAD gene orders are located at the GAL1 promoters downstream of pYES2 carriers
Utilize galactolipin inducible gene expression.
3) use the electric-shocking method of electroporation apparatus that the expression vector pY- Δs 9FAD of recombination is successfully transformed into yeast
In BY4389.It is also His that oleic acid, which synthesizes deficiency ole1 mutant strain yeast BY4389,–、Leu–、Ura–Auxotrophic strain,
After being transferred to pY- Δ 9FAD plasmids, because pYES2 carriers carry the URA3 genes of coding uracil, transgenic yeast thus can be
It is grown on the synthetic media (SC-U) of uracil-deficient in order to screen.
4) for transgenic yeast after galactolipin Fiber differentiation, GC-MS detects the aliphatic acid of yeast esterification.As a result (Fig. 4)
Show that palmitic acid and stearic acid desaturation can be respectively palmitoleic acid and oleic acid in yeast by Δ 9FAD coded by said gene albumen.
Mass spectrum (B and C in Fig. 4) is the results show that the unsaturated bond on palmitoleic acid and oleic acid is all located on 9 carbon location of Δ.Show from scarce
The protein for carving the coded by said gene being cloned into edge green alga has the function of 9 fatty acid desaturase of Δ.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (9)
1. a kind of polypeptide, which is characterized in that 62-432 of the amino acid sequence of the polypeptide such as SEQ ID NO.15 or
Shown in SEQ ID NO.15.
2. a kind of nucleotide of separation, which is characterized in that the sequence of the nucleotide is：
A) 158bp to the 1st 453bp, SEQ ID of the 341bp to the 1st 453bp, SEQ ID NO.3 of SEQ ID NO.3
Nucleotide sequence shown in NO.3 or SEQ ID NO.12；Or
B) nucleotide sequence of nucleotide sequence complete complementary shown in and a).
3. a kind of recombinant expression carrier, which is characterized in that the recombinant expression carrier is by the nucleotide described in claim 2
With the recombinant expression carrier constructed by plasmid or virus.
4. recombinant expression carrier according to claim 3, which is characterized in that the plasmid is pYES2 plasmids.
5. a kind of genetically engineered host cell, which is characterized in that the host cell is selected from one of the following：
A) host cell of the nucleotide conversion or transduction described in claim 2 is used；
B) host cell of the recombinant expression carrier conversion or transduction described in claim 3 is used.
6. host cell according to claim 5, which is characterized in that the host cell is that bacterial cell, fungi are thin
Born of the same parents, plant cell or zooblast.
7. host cell according to claim 6, which is characterized in that the host cell is yeast cells.
8. the recombinant expression described in the nucleotide, claim 3 or 4 described in polypeptide described in claim 1, claim 2 carries
The purposes of body or any host cells of claim 5-7 in synthesizing polyunsaturated fatty acid.
9. the recombinant expression described in the nucleotide, claim 3 or 4 described in polypeptide described in claim 1, claim 2 carries
Palmitic acid desaturation is being palmitoleic acid or by stearic acid desaturation by body or any host cells of claim 5-7
For the purposes in oleic acid.
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Non-Patent Citations (3)
| Title |
|---|
| Identification and Functional Expression of a △9-Fatty Acid Desaturase from Psychrobacter urativorans in Escherichia coli;Yan Li et al.;《Lipids》;20080207;第43卷;第207-213页 * |
| 缺刻缘绿藻ω3脂肪酸去饱和酶基因(ω3FAD)在酿酒酵母中的低温诱导表达;李慧 等;《农业生物技术学报》;20121231;第20卷(第7期);第735-744页 * |
| 缺刻缘绿藻转录组测序及脂质代谢相关基因注释;陈思弘 等;《上海海洋大学学报》;20120930;第21卷(第5期);第662-670页 * |
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